CN111699003A - anti-LRP 5/6 antibodies and methods of use - Google Patents

Abstract

The present invention provides anti-LRP 5/6 monoclonal antibodies and related compositions that can be used in any of a variety of therapeutic methods for the treatment of diseases and disorders associated with Wnt pathway signaling.

Description

anti-LRP 5/6 antibodies and methods of use

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 62/607,879 filed on 12/19/2017 and U.S. provisional application No. 62/680,515 filed on 6/4/2018, both of which are incorporated herein by reference in their entirety.

Statement regarding sequence listing

The sequence listing associated with this application is provided in textual format in lieu of a paper copy and is incorporated by reference herein. The text file containing the sequence listing is named SRZN _005_02WO _ st25. txt. The text file was 181KB, created 12 months and 19 days 2018 and submitted electronically via EFS-Web.

Technical Field

The invention relates generally to anti-LRP 5/6 antibodies, compositions, and methods of use of the anti-LRP 5/6 antibodies, compositions. Such antibodies are useful, for example, in methods of modulating the Wnt signaling pathway.

Background

Wnt ("wingless associated integration site" or "wingless and Int-1" or "wingless-Int") ligands and their signals play a key role in controlling the development, homeostasis and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, breast, taste bud, ovary, cochlea and many others (e.g., reviewed by Clevers, Loh and Nusse, 2014; 346:1table 1B 8012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue damage.

One of the therapeutic challenges in modulating Wnt signaling is the existence of multiple Wnt ligands and Wnt receptors, frizzled 1-10(Fzd1-10), where many tissues express multiple Fzd and overlapping Fzd. The canonical Wnt signals also involve Low Density Lipoprotein (LDL) receptor-associated protein 5(LRP5) or Low Density Lipoprotein (LDL) receptor-associated protein 6(LRP6) as co-receptors, which are widely expressed in various tissues in addition to Fzd. Thus, there is a clear need in the art for binding moieties, such as antibodies, that specifically bind to one or more of Fzd, LRP5 or LRP 6. The present invention addresses this need.

Disclosure of Invention

An isolated antibody or antigen binding fragment thereof that binds to one or more LRP5 or LRP6 receptors, comprising a sequence comprising: CDRH1, CDRH2 and CDRH3 sequences as shown for any of the antibodies of table 1A; and/or CDRL1, CDRL2, and CDRL3 sequences as set forth for any of the antibodies of table 1A, or a variant of said antibody or antigen binding fragment thereof, said variant comprising one or more amino acid modifications, wherein said variant comprises fewer than 8 amino acid substitutions in said CDR sequences. In particular embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs 1-24; or a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NOs 1-24.

In particular embodiments, any one of the antibodies or antigen-binding fragments thereof is humanized. In certain embodiments, any one of the antibodies or antigen-binding fragments thereof is a single chain antibody, scFv, monovalent antibody lacking a hinge region, VHH, or single domain antibody (sdAb), or minibody. In particular embodiments, any one of the antibodies or antigen-binding fragments thereof is a VHH or sdAb. In particular embodiments, any one of the antibodies or antigen-binding fragments thereof is a Fab or Fab' fragment.

In certain embodiments, any one of the antibodies or antigen-binding fragments thereof is a fusion protein. In certain embodiments, the antibody or antigen-binding fragment thereof is fused to a polypeptide sequence that binds one or more Fzd receptors. In certain embodiments, the polypeptide sequence that binds one or more Fzd receptors is an antibody or antigen-binding fragment thereof that binds one or more Fzd receptors.

In certain embodiments, any of the isolated antibodies or antigen binding fragments thereof disclosed herein bind to LRP5, LRP6, or both LRP5 and LRP 6.

In related embodiments, the present disclosure provides an isolated antibody or antigen binding fragment thereof that competes for binding to LRP5 or LRP6 with any one of the antibodies disclosed herein.

In particular embodiments, any one of the antibodies or antigen-binding fragments thereof binds to LRP5 or LRP6 with a KD of 50 μ Μ or less.

In particular embodiments, any one of the antibodies or antigen-binding fragments thereof modulates the Wnt signaling pathway in a cell, optionally a mammalian cell. In particular embodiments, any one of the antibodies or antigen-binding fragments thereof increases signaling through the Wnt signaling pathway in the cell. In particular embodiments, any one of the antibodies or antigen-binding fragments thereof reduces signaling through the Wnt signaling pathway in the cell. In certain embodiments, the Wnt signaling pathway is a canonical Wnt signaling pathway or an atypical Wnt signaling pathway.

In further related embodiments, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the present disclosure provides an expression vector comprising the isolated polynucleotide and an isolated host cell comprising the expression vector.

In another embodiment, the present disclosure provides a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier and a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof disclosed herein.

In additional embodiments, the present disclosure provides a method for agonizing a Wnt signaling pathway in a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof disclosed herein that increases Wnt signaling. In particular embodiments, the antibody or antigen-binding fragment thereof is a fusion protein comprising a polypeptide sequence that binds to one or more frizzled (Fzd) receptors.

In another embodiment, the present disclosure provides a method for inhibiting a Wnt signaling pathway in a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof disclosed herein that inhibits Wnt signaling.

In another embodiment, the disclosure includes a method for treating a subject having a disease or disorder associated with reduced Wnt signaling, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof disclosed herein that is an agonist of the Wnt signaling pathway. In particular embodiments, the disease or disorder is selected from the group consisting of: bone fracture, stress fracture, vertebral compression fracture, osteoarthritis, osteoporosis, osteoporotic fracture, nonunion fracture, delayed union fracture, spinal fusion, preoperative optimization of spinal surgery, osteonecrosis, osteointegration of implants or orthopedic devices, osteogenesis imperfecta, bone graft, tendon repair, tendon-bone integration, tooth growth and regeneration, maxillofacial surgery, dental implant, periodontal disease, maxillofacial reconstruction, jaw, hip, or femoral head necrosis, avascular necrosis, hair loss, hearing loss, vestibular dysfunction, macular degeneration, age-related macular degeneration (AMD), vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs 'dystrophe, corneal diseases, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, sarcopenia and cachexia, diseases affecting the Blood Brain Barrier (BBB), spinal cord injury, spinal cord disease, oral mucositis, short bowel syndrome, Inflammatory Bowel Disease (IBD), metabolic syndrome, diabetes, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency, wound healing, diabetic foot ulcer, bedsores, venous leg ulcer (venous leg ulcer), bullous epidermal lysis, skin dysplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell disorders, immunodeficiency, graft-versus-host disease, acute kidney injury, chronic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis, acute liver failure of all causes, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of all causes, cirrhosis, hepatic fibrosis of all causes, chronic lung fibrosis of all causes, and/or chronic kidney disease of all causes, Portal hypertension, chronic liver insufficiency of all causes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis c virus-induced liver disease (HCV), hepatitis b virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis a virus-induced liver disease (HAV) and hepatitis delta virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small liver (for size)" syndrome in liver surgery and transplantation, congenital liver disease and disorder, any other liver disorder or disease resulting from genetic disease, degeneration, aging, drug or injury.

In related embodiments, the present disclosure provides a method for treating a subject having a disease or disorder associated with increased or enhanced Wnt signaling, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof disclosed herein that is an inhibitor of the Wnt signaling pathway. In certain embodiments, the disease or disorder is selected from the group consisting of: tumors and cancers, degenerative disorders, fibrosis, heart failure, coronary artery disease, ectopic ossification, osteopetrosis and congenital high bone mass disorders.

Drawings

FIG. 1 shows a graphical representation of the crystal structure of LRP6E3E4: VHH26 binding complex. LRP6E3E4 is shown in transparent gray and VHH26 is shown in dark gray. The positions of the CDR loops of VHH26 are labeled. Glycans on the surface of LRP6E3E4 are shown in bar representation.

FIG. 2 shows a graphical representation of the crystal structure of LRP6E3E4: VHH36 binding complex. LRP6E3E4 is shown in transparent gray, and VHH36 is shown in dark gray. The positions of the CDR loops of VHH36 are labeled. Glycans on the surface of LRP6E3E4 are shown in bar representation.

Figure 3A shows Wnt activation using a375 Wnt reporter assay. Figure 3B shows Wnt reporter activation in HEK293 cells with addition of Rspo. The clones tested in both assays were Wnt-replacement molecules comprising Fzd conjugate 18R5 scFv combined with LRP VHH or sdAb conjugate. The FLAG and His tag were attached to the 18R5: LRP replacement molecule.

Detailed Description

The present disclosure relates to antibodies and antigen binding fragments thereof that specifically bind to LRP5 and/or LRP6, particularly antibodies having specific and functional properties of the specific LRP receptor. One embodiment of the invention encompasses specific humanized antibodies and fragments thereof that are capable of binding to LRP5 and/or LRP6 and modulating downstream Wnt pathway signaling and biological effects. For convenience, the term "anti-LRP 5/6" is used to collectively refer to antibodies and antigen-binding fragments thereof that bind to either or both of LRP5 and/or LRP 6.

Embodiments of the invention relate to the use of anti-LRP 5/6 antibodies or antigen binding fragments thereof in the diagnosis, assessment, and treatment of diseases and disorders associated with the Wnt signaling pathway. In certain embodiments, the subject antibodies are used to treat or prevent diseases and disorders associated with aberrant (e.g., increased or decreased) Wnt signaling or for which decreasing or increasing Wnt signaling would provide a therapeutic benefit.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the scope of the art, many of which are described below for purposes of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.) (2009); ausubel et al, eds molecular Biology laboratory guidelines (Short Protocols in molecular Biology), 3 rd edition, Willd's father, 1995; sambrook and Russell, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual) (3 rd edition, 2001); maniatis et al molecular cloning: a laboratory Manual (1982); cloning of DNA: practical methods (DNA Cloning: A Practical Approach), volumes I and II (edited by Glover); oligonucleotide Synthesis (oligo Synthesis) (n. goal editions, 1984); nucleic Acid Hybridization (Nucleic Acid Hybridization), edited by hames and s.higgins, 1985; transcription and Translation (b.hames and s.higgins editions, 1984); animal Cell Culture (Animal Cell Culture), edited by r.freshney, 1986; perbal, practical guidelines for Molecular Cloning (1984), and other similar references.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Unless explicitly stated otherwise, each embodiment in this specification applies to all other embodiments mutatis mutandis.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions, or as commonly practiced in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless a specific definition is provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques may be used for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

Embodiments of the invention relate to antibodies that bind to LRP5 and/or LRP 6. 1-24 show the sequences of illustrative antibodies or antigen-binding fragments thereof or Complementarity Determining Regions (CDRs) thereof.

As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab ', F (ab')₂Fv), single chain (scFv), VHH or sdAb (also referred to as nanobody), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment with a desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) with a desired specificity. "binary" multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; P.Holliger et al, Proc. Natl. Acad. Sci. USA, 906444-6448, 1993) are also particular forms of antibodies contemplated herein. Also included herein are polypeptides comprising s linked to a CH3 domain The mini-body of cFv (S.Hu et al, Cancer Res., 56,3055-3061, 1996). See, e.g., Ward, E.S. et al, Nature 341,544-546 (1989); bird et al, Science 242,423-426, 1988; huston et al, Proc. Natl. Acad. Sci. USA (PNAS USA), 85, 5879-; PCT/US 92/09965; WO 94/13804; holliger et al, Proc. Natl. Acad. Sci. USA 906444-6448, 1993; reiter et al, Nature Biotech, 14,1239-1245, 1996; hu et al, cancer research, 56,3055 and 3061, 1996.

The term "antigen-binding fragment" as used herein refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and/or light chain that binds to an antigen of interest, in particular LRP5 and/or LRP 6. In this regard, an antigen-binding fragment of an antibody described herein can include 1, 2, 3, 4, 5, or all 6 CDRs from the VH and VL sequences of an antibody that binds LRP5 and/or LRP6 shown herein. Antigen binding fragments of LRP5/6 specific antibodies described herein are capable of binding to LRP5 and/or LRP 6. In certain embodiments, the antigen-binding fragment or antibody comprising the antigen-binding fragment increases a Wnt signaling event. In certain embodiments, the antibody or antigen-binding fragment specifically binds to and/or modulates a biological activity of a human Wnt signaling pathway. In certain embodiments, the antibody or antigen-binding fragment thereof increases or decreases Wnt signaling.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antibody, and that is otherwise capable of being used in an animal to produce an antibody that is capable of binding to an epitope of the antigen. An antigen may have one or more epitopes. In certain embodiments, an antibody is considered to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In certain embodiments, when the equilibrium dissociation constant is ≦ 10^-7Or 10^-8M, the antibody is believed to bind specifically to the antigen. In some embodiments, the equilibrium dissociation constant may be ≦ 10^-9M is equal to or less than 10^-10M。

The term "epitope" encompasses peptidesAny determinant, preferably a polypeptide determinant, that specifically binds to an immunoglobulin or T cell receptor. An epitope is the region of an antigen that is bound by an antibody. In certain embodiments, an epitope determinant comprises a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments may have a particular three-dimensional structural characteristic and/or a particular charge characteristic. In certain embodiments, an antibody is considered to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. When the equilibrium dissociation constant is less than or equal to 10 ^-7Or 10^-8M, the antibody is believed to bind specifically to the antigen. In some embodiments, the equilibrium dissociation constant may be ≦ 10^-9M is equal to or less than 10^-10M。

In certain embodiments, the antibodies and antigen-binding fragments thereof as described herein comprise sets of heavy and light chain CDRs inserted between sets of heavy and light chain Framework Regions (FRs), respectively, the FR sets providing support for the CDRs and defining the spatial relationship of the CDRs relative to each other. As used herein, the term "set of CDRs" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are denoted "CDR 1", "CDR 2" and "CDR 3", respectively. Thus, the antigen binding site comprises six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Polypeptides comprising a single CDR (e.g., CDR1, CDR2, or CDR3) are referred to herein as "molecular recognition units". Crystallographic analysis of various antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the antigen of the heavy chain CDR 3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

As used herein, the term "FR set" refers to the four flanking amino acid sequences of the CDRs in the set of CDRs that frame the heavy or light chain V regions. Some FR residues may contact the bound antigen; however, the FR is primarily responsible for folding the V region into an antigen binding site, particularly FR residues immediately adjacent to the CDRs. Within the FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop having about 90 amino acid residues. When the V region folds into a binding site, the CDRs are displayed as prominent loop motifs that form the antigen binding surface. It is generally recognized that there are conserved structural regions of the FR that change the folded shape of the CDR loops into certain "canonical" structures-regardless of the exact CDR amino acid sequence. Further, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction of the heavy and light chains of antibodies.

The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, e.a. et al, Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), 4 th edition, american Department of Health and Human Services (US Department of Health and Human Services), 1987 and updates thereof, currently available on the internet (immune. Alternatively, the term "can be used by using what is available under http:// www.imgt.org"

(International ImmunoGeneTiCs information System

) "to determine CDRs (see, e.g., Lefranc, M. -P. et al (1999) Nucleic Acids research (Nucleic Acids Res.), 27: 209-212; ruiz, M. et al (2000) nucleic acids research, 28: 219-221; lefranc, M. -P. (2001) nucleic acids research, 29: 207-; lefranc, M. -P. (2003) nucleic acids research 31:307- & 310; lefranc, M. -P. et al (2004) computer biology (In silica Biol.), 5,0006[ Epub ]],5:45-60(2005)](ii) a Lefranc, M. -P. et al (2005) nucleic acids research 33: D593-597; lefranc, M. -P. et al (2009) nucleic acids research 37: D1006-1012; lefranc, M. -P. et al (2015) nucleic acids research, 43: D413-422).

"monoclonal antibody" refers to a homogeneous population of antibodies, wherein the monoclonal antibodies comprise the amino acids (both naturally occurring and non-naturally occurring) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact single antibodies Cloned and full-length monoclonal antibodies, and fragments thereof (e.g., Fab ', F (ab')₂Fv), single chain (scFv), variants thereof, fusion proteins comprising an antigen binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that includes an antigen binding fragment (epitope recognition site) having a desired specificity and the ability to bind an epitope. There is no intention to be limited as to the source of the antibody or the manner in which it is formed (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, etc.). The term encompasses whole immunoglobulins as well as fragments and the like described above under the definition of "antibody".

The proteolytic enzyme papain preferentially cleaves IgG molecules to produce fragments, two of which (f (ab) fragments) each comprise a covalent heterodimer comprising an intact antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide fragments comprising F (ab')₂And (3) fragment. Fv fragments for use in accordance with certain embodiments of the invention can be produced by preferential proteolytic cleavage of IgM, and in rare cases by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. The Fv fragment comprises a non-covalent V _H::V_LA heterodimer comprising an antigen binding site that retains many of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al (1972) Proc. Natl. Acad. Sci. USA 69: 2659-; hochman et al (1976) biochemistry (Biochem) 15: 2706-2710; and Ehrlich et al (1980) biochemistry 19: 4091-.

In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, the Kappa body (Ill et al, protein engineering (prot. eng.) 10:949-57(1997), the minibody (Martin et al, J.Eur. Biol.Biol.Proc. (EMBO J.) 13:5305-9 (1994)), the diabody (Holliger et al, Proc. Natl. Acad. Sci. USA 90:6444-8(1993), or Janusins (Trauecker et al, J.Eur. Biol.Biol.Proc. 10:3655-59(1991) and Trauecker et al, J.Cancer Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques following the teachings of this application for selecting antibodies with the desired specificity A bispecific antibody in which the domain specifically binds to a second molecule. These antibodies may be produced by recombinant molecular biology techniques or may be physically conjugated together.

Single chain fv (scFv) polypeptides are covalently linked V_H::V_LHeterodimers according to comprising V linked by a peptide-encoding linker_HAnd V_LThe gene of the coding gene is expressed by fusion. Huston et al (1988) Proc. Natl. Acad. Sci. USA 85(16) 5879-. Various methods have been described to distinguish the chemical structures used to convert naturally polymerized but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into three-dimensional structures substantially similar to those of the antigen binding site. See, e.g., U.S. Pat. nos. 5,091,513 and 5,132,405 to Huston et al; and U.S. Pat. No. 4,946,778 to Ladner et al.

In certain embodiments, an LRP5/6 binding antibody as described herein takes the form of a dimer. A dimer is a multimer of polypeptides, each polypeptide comprising a first domain that comprises a binding region of an immunoglobulin light chain and a second domain that comprises a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: an antigen binding site is formed by the association of a first domain of one polypeptide with a second domain of another polypeptide in a polymer (WO 94/13804).

dAb fragments of antibodies consist of VH domains (Ward, E.S. et al, Nature 341,544-546 (1989)).

In the case of bispecific antibodies, these may be conventional bispecific antibodies, which may be manufactured in various ways (Holliger, P. and Winter G., (Current opinion Biotechnol.), (4, 446-449(1993)), e.g.chemically or from hybrid hybridomas, or may be bispecific antibody fragments of any of the above-mentioned bispecific antibody fragments. Diabodies and scFvs can be constructed without an Fc region using only variable domains, potentially reducing the effect of anti-idiotypic reactions.

In contrast to bispecific whole antibodies, bispecific diabodies can also be particularly useful, since they can be easily constructed and expressed in e. Phage display (WO94/13804) can be used to easily select diabodies (and many other polypeptides, such as antibody fragments) from libraries with appropriate binding specificity. If one arm of the diabody is kept constant, e.g.with specificity for antigen X, a library can be generated in which the other arm is varied and antibodies with the appropriate specificity selected. Bispecific whole antibodies can be generated by knob-and-hole (J.B.Ridgeway et al, protein engineering, 9, 616-.

In certain embodiments, the antibodies described herein can be administered to a subject in need thereof

The method is provided in a form.

Is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, the Netherlands; see also, for example, US 20090226421). This patented antibody technology produces a stable smaller antibody form with a longer therapeutic window than current small antibody forms would expect. The IgG4 antibody is considered inert and therefore does not interact with the immune system. The whole human IgG4 antibody can be modified by eliminating the hinge region of the antibody to obtain a half-molecule fragment (GenMab, Utrecht) with different stability properties compared to the corresponding whole IgG 4. Bisecting the IgG4 molecule leaves only one region available for binding to a cognate antigen (e.g., a disease target)) Is/are as follows

To is and

and therefore binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding may not stimulate cancer cells to grow as can be seen using a bivalent antibody with the same antigen specificity, and thus

The technology may provide a treatment option for some types of cancer that may be refractory to treatment with conventional antibodies. When treating some forms of cancer, the patient is,

may be of great benefit, allowing better distribution of molecules over larger solid tumors and potentially increased efficacy.

In certain embodiments, the antibodies of the disclosure can take the form of a VHH or sdAb. VHH or sdAb is encoded by a single gene and is produced efficiently in almost all prokaryotic and eukaryotic hosts, such as e.g. escherichia coli (see e.g. us patent No. 6,765,087), moulds (e.g. Aspergillus (Aspergillus) or Trichoderma (Trichoderma)) and yeasts (e.g. Saccharomyces (Saccharomyces), kluyveromyces (kluyveromyces), Hansenula (Hansenula) or Pichia (Pichia)) (see e.g. us patent No. 6,838,254). The production process is scalable and already generates several kilogram quantities of VHH or sdAb. The VHH or sdAb can be formulated as a ready-to-use solution with a long shelf life.

The method (see e.g. WO 06/079372) is a patented method for the generation of VHH or sdAb against a desired target based on automated high-throughput selection of B-cells.

In certain embodiments, the anti-LRP 5/6 antibodies or antigen binding fragments thereof disclosed herein are humanized. This involves chimeric molecules, typically prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise the entire variable domain fused to the constant domain or only the CDRs grafted onto the appropriate framework regions in the variable domain. The epitope binding site can be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen for human individuals, but the possibility of an immune response to foreign variable regions still exists (LoBuglio, A.F. et al, (1989) Proc. Natl. Acad. Sci. USA 86: 4220-4224; Queen et al, Proc. Natl. Acad. Sci. USA (1988)86: 10029-10033; Riechmann et al, Nature (1988)332: 323-327). An illustrative method for humanizing the anti-LRP 5/6 antibodies disclosed herein comprises the method described in U.S. patent No. 7,462,697.

Another approach has focused not only on providing human-derived constant regions, but also on modifying the variable regions so as to remodel them into a form as close to human as possible. The variable regions of the heavy and light chains are known to contain three Complementarity Determining Regions (CDRs) that vary in response to the epitope in question and determine binding capacity, flanked by four Framework Regions (FRs) that are relatively conserved in a given species and putatively provide a scaffold for the CDRs. When a non-human antibody is prepared with respect to a particular epitope, the variable region can be "reshaped" or "humanized" by grafting CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. This method has been reported to be applied to various antibodies by: sato, K. et al, (1993) cancer research 53:851-856.Riechmann, L., et al, (1988) Nature 332: 323-327; verhoeyen, M., et al, (1988) science 239: 1534-1536; kettleborough, C.A., et al, (1991) protein engineering 4: 773-; maeda, H, et al, (1991) human antibody hybridoma 2: 124-; gorman, S.D., et al, (1991) Proc. Natl. Acad. Sci. USA 88: 4181-4185; tempest, P.R., et al, (1991) Bio/Technology (Bio/Technology) 9: 266-271; co, M.S., et al, (1991) Proc. Natl. Acad. Sci. USA 88: 2869-; carter, P.et al, (1992) Proc. Natl. Acad. Sci. USA 89: 4285-; and Co, M.S. et al, (1992) J Immunol 148: 1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody, also referred to as one or more CDRs that are "derived" from the one or more CDRs of the original antibody.

In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, chimeric antibodies include antigen-binding fragments of the anti-LRP 5/6 antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class derived from the parent antibody, comprising IgA (comprising subclasses IgA1 and IgA2), IgD, IgE, IgG (comprising subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. In further embodiments, the heterologous Fc domain may comprise CH2 and CH3 domains from one or more of the different Ig classes. As described above with respect to humanized antibodies, an anti-LRP 5/6 antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of an antibody described herein), or may include the entire variable domain (VL, VH, or both).

In certain embodiments, the Fc region of an antibody or fragment thereof may be derived from any of a variety of different Fc's, including but not limited to wild-type or modified IgG1, IgG2, IgG3, IgG4, or other isotypes, such as wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (including mutations in the core hinge region that prevent formation of IgG4 half molecules), human IgA, human IgE, human IgM, or modified IgG1 known as IgG1 lalapc. The L235A, P329G (LALA-PG) variants have been shown to abrogate complement binding and fixation and Fc- γ dependent antibody dependent cell mediated cytotoxicity (ADCC) in both murine IgG2a and human IgG 1. In particular embodiments of any of the iggs disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A or P236G.

In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise fusion proteins, e.g., Wnt signaling pathway agonist fusion proteins, also referred to herein as "Wnt substitutes". The Wnt substitutes of the invention are generally biologically active in binding to the cognate frizzled receptor and in activation of Wnt signaling, i.e., the substitutes are Wnt agonists. The term "Wnt agonist activity" refers to the ability of an agonist to mimic the effect or activity of Wnt protein binding to frizzled. The ability of the agonists of the invention to mimic Wnt activity can be demonstrated by a variety of assays. Agonists of the invention typically elicit a response or activity similar to or identical to that elicited by the natural ligand of the receptor. In particular, the agonists of the invention enhance the canonical Wnt/β -catenin signaling pathway. As used herein, the term "enhance" refers to a measurable increase in the level of Wnt/β -catenin signaling compared to the level in the absence of an agonist of the invention.

In particular embodiments, Wnt signaling pathway agonist fusion proteins (or Wnt substitutes) include an anti-LRP 5/6 antibody or antigen-binding fragment thereof disclosed herein fused to a polypeptide that specifically binds one or more frizzled (Fzd) receptors. In particular embodiments, the polypeptide that specifically binds to one or more Fzd receptors is an antibody or antigen-binding fragment thereof. In certain embodiments, it is an antibody or antigen-binding fragment thereof disclosed in U.S. provisional patent application No. 62/607,877 entitled "Anti-Frizzled antibodies and Methods of Use", attorney docket No. SRZN-004/00US, filed on 19.12.2017, which is incorporated herein by reference in its entirety.

In certain embodiments, the Fzd binding domain can be selected fromSelf-affinity, e.g. at least about 1 × 10^-7M, at least about 1 × 10^-8M, at least about 1 × 10^-9M or at least about 1 × 10^-10KD of M binds to any domain of Fzd. Suitable Fzd binding domains include, but are not limited to: de novo designed Fzd binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind to one or more Fzd proteins; a VHH or sdAb derived binding domain; knottin (knottin) -based engineered scaffolds; norrin and engineered binding fragments derived therefrom, naturally occurring Fzd binding domains, and the like. The Fzd binding domains can be affinity selected to enhance binding to a desired Fzd protein or proteins, for example to provide tissue selectivity.

In some embodiments, the Fzd binding domain binds to one, two, three, four, five or more different frizzled proteins, for example one or more human frizzled proteins of human frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd 10. In some embodiments, the Fzd binding domain binds to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd 8. In other embodiments, the Fzd binding domain is selective for one or more frizzled proteins of interest, e.g., has at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or more specificity for the one or more desired frizzled proteins relative to other frizzled proteins.

In certain embodiments, the Fzd binding domain comprises the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vantictumab). In certain embodiments, the Fzd binding domain is a scFv comprising the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vantictumab). See, for example, U.S. patent No. 8507442, which is specifically incorporated herein by reference. For example, the CDR sequences of OMP-18R5 comprise: heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:25), heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:26) and heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 27); and (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:28) or SGDNIGSFYVH (SEQ ID NO:31), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO:29) or DKSNRRPSG (SEQ ID NO:32), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:30) or QSYANTLSL (SEQ ID NO: 33). In particular embodiments, the frizzled binding domain is an antibody or derivative thereof, including but not limited to scFv, minibodies, VHH, or sdAb, as well as various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences comprise one or more amino acid modifications.

In other embodiments, the Fzd binding domain comprises a variable region sequence or CDRs thereof from any one of a variety of frizzled-specific antibodies known in the art and commercially available or can be produced de novo. Any of the frizzled polypeptides can be used as an immunogen or in a screening assay for the development of antibodies. Non-limiting examples of frizzled binding domains include antibodies commercially available from Biolegend, e.g., clone CH3A4a7 specific for human frizzled 4(CD344), clone W3C4E11 specific for human Fz9(CD 349); antibodies available from ebola (Abcam), e.g., ab64636 specific for Fz 7; ab83042 specific for human Fz 4; ab77379 specific for human Fz 7; ab75235 specific for human Fz 8; ab102956 specific for human Fz 9; and the like. Other examples of suitable antibodies are described in, inter alia, U.S. patent applications 20140105917; U.S. patent application 20130230521; U.S. patent application 20080267955; U.S. patent application 20080038272; U.S. patent application 20030044409; and the like, each of which is specifically incorporated herein by reference.

The Fzd-binding portion of the surrogate may be an engineered protein selected for structural homology to the frizzled-binding region of the Wnt protein. Such proteins can be identified by screening structural databases for homology. Thus, the original protein, for example, the microbial Bh1478 protein, was identified. The native protein is then engineered to provide increased affinity amino acid substitutions, and further selection can be made by affinity maturation to have increased affinity and selectivity upon binding to the desired frizzled protein. Non-limiting examples of frizzled binding moieties include Fz27 and Fz27-B12 proteins.

The anti-LRP 5/6 antibody or antigen-binding fragment thereof and Fzd binding domain may be directly linked or may be separated by a linker (e.g., a polypeptide linker or a non-peptide linker, etc.). The region of the Wnt substitute that binds one or more Fzd receptors and the region of the Wnt substitute that binds LRP5 and/or LRP6 may be contiguous or separated by a linker (e.g., a polypeptide linker or a non-peptide linker, etc.). The length of the linker, and thus the spacing between the binding domains, can be used to modulate signal strength and can be selected according to the intended use of the Wnt substitute. The reinforcement distance between binding domains may vary, but in certain embodiments may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms. In some embodiments, the joint is a rigid joint, and in other embodiments, the joint is a flexible joint. Where the linker is a peptide linker, the linker may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids in length and have sufficient length and amino acid composition to enhance the distance between the binding domains. In some embodiments, the linker comprises or consists of one or more glycine and/or serine residues.

Wnt substitutes can be multimerized, for example, by Fc domains, by means of cascades, coiled coils, polypeptide zippers, biotin/avidin or streptavidin multimerization, and the like. Wnt substitutes can also be linked to moieties known in the art such as PEG, Fc, etc. to enhance in vivo stability.

In certain embodiments, the Wnt substitutes directly activate canonical Wnt signaling by binding to one or more Fzd proteins and LRP5 and/or LRP6, particularly by binding to these proteins on the surface of a cell (e.g., the surface of a human cell). Direct activation of Wnt signaling by Wnt substitutes is in contrast to potentiation of Wnt signaling, which only enhances activity in the presence of native Wnt proteins.

The present Wnt substitutes activate Wnt signaling, for example, by mimicking the effect or activity of Wnt protein binding to frizzled. The ability of a Wnt substitute of the invention to mimic Wnt activity can be demonstrated by a variety of assays. Wnt substitutes typically elicit responses or activities that are similar or identical to those elicited by the natural ligand of the receptor. In particular, the Wnt substitutes of the invention enhance the canonical Wnt/β -catenin signaling pathway. As used herein, the term "potentiation" refers to a measurable increase in the level of Wnt/β -catenin signaling compared to the level in the absence of a Wnt substitute of the invention.

In certain embodiments, an antibody or antigen-binding fragment thereof disclosed herein inhibits Wnt pathway signaling. In particular embodiments, binding of an anti-LRP 5/6 antibody or antigen binding fragment thereof blocks or inhibits endogenous Wnt binding to one or more LRP5/6 receptors on the surface of a cell, thereby reducing or inhibiting Wnt signaling.

Various methods are known in the art for measuring the level of canonical Wnt/β -catenin signaling. These methods include, but are not limited to, assays that measure: wnt/β -catenin target gene expression; TCF reporter gene expression; beta-catenin stabilization; LRP phosphorylation; axin translocates from the cytoplasm to the cell membrane and binds LRP. The canonical Wnt/β -catenin signaling pathway ultimately causes changes in gene expression by the transcription factors TCF7, TCF7L1, TCF7L2, and LEF. Transcriptional responses to Wnt activation have been characterized in many cells and tissues. Thus, global transcription profiling by methods well known in the art can be used to assess Wnt/β -catenin signaling activation or inhibition.

Changes in Wnt-responsive gene expression are typically mediated by TCF and LEF transcription factors. The TCF reporter assay assesses transcriptional changes in the TCF/LEF control genes to determine the level of Wnt/β -catenin signaling. The TCF reporter assay was first described by Koriek, V. et al, 1997. This method, also known as TOP/FOP, involves the use of three copies of the optimal TCF motif CCTTTGATC or three copies of the mutation motif CCTTTGGCC (ptopfofi _ ASH and pFOPFI _ ASH, respectively) located upstream of the minimal c-Fos promoter driving luciferase expression to determine the transactivation activity of endogenous p-catenin/TCF 4. A higher ratio of the activities of the two reporters (TOP/FOP) indicates higher β -catenin/TCF 4 activity, while a lower ratio of the activities of the two reporters indicates lower β -catenin/TCF 4 activity.

Various other reporter transgenes that respond to Wnt signaling are present intact in the animal and therefore effectively reflect endogenous Wnt signaling. These reporters are based on a multimeric TCF binding site that drives expression of LacZ or GFP, which can be readily detected by methods known in the art. These reporter genes include: TOP-GAL, BAT-GAL, ins-TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tm1(cre/ERT2), TOPdGFP.

In some cases, the recruitment of dephosphorylated β -catenin to the membrane, its stabilization and phosphorylation state, and its translocation to the nucleus, mediated by complexes with TCF transcription factors and TNIK, are key steps in the Wnt signaling pathway (klapuloz-Brown Z et al, journal of public science library, 2(9) e945,2007). Stabilization is mediated by dishevelled family proteins that inhibit the "disruption" of the complex, thus reducing the degradation of intracellular β -catenin, and thereafter translocation of β -catenin to the nucleus. Thus, measuring the level and location of β -catenin in a cell reflects well the level of Wnt/β -catenin signaling. A non-limiting example of such an assay is the "Biolmage β -catenin redistribution assay" (Thermo Scientific) which provides recombinant U20S cells stably expressing human β -catenin fused to the C-terminus of Enhanced Green Fluorescent Protein (EGFP). Imaging and analysis were performed using a fluorescence microscope or HCS platform, allowing visualization of the level and distribution of EGFP- β -catenin.

Another way to inhibit disruption of the complex is to remove Axin by recruiting Axin to the cytoplasmic tail of the Wnt co-receptor LRP. Axin has been shown to preferentially bind to the phosphorylated form of the LRP tail region. Thus, for example, visualization of Axin translocation with GFP-Axin fusion proteins is another method for assessing Wnt/β -catenin signaling levels.

In certain embodiments, a Wnt signaling pathway agonist enhances or increases canonical Wnt pathway signaling (e.g., β -catenin signaling) by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400%, or 500% compared to neutral-induced β -catenin signaling or a negative control, as measured in an assay described above, e.g., in a TOPFIash assay. Negative controls may be included in these assays. In particular embodiments, a Wnt agonist can enhance β -catenin signaling by 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or more, as measured in the above-described assays, e.g., in the TOPFIash assay or any of the other assays mentioned herein, compared to activity in the absence of the agonist.

In certain embodiments, the Wnt signaling pathway antagonist or inhibitor inhibits or reduces canonical Wnt pathway signaling (e.g., β -catenin signaling) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100% compared to β -catenin signaling or a negative control observed in the presence of a neutral substance, as measured in the above-described assays, e.g., in the TOPFIash assay. Positive controls may be included in these assays.

As used herein, "Wnt gene product" or "Wnt polypeptide" encompasses native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments, and chimeric Wnt polypeptides. In particular embodiments, the Wnt polypeptide is a native human full length mature Wnt protein.

For example, human native sequence Wnt proteins of interest in this application comprise the following: wnt-1(GenBank accession NM-005430); wnt-2(GenBank accession NM-003391); Wnt-2B (Wnt-13) (GenBank accession No. NM-004185 (isoform 1), NM-024494.2 (isoform 2)), Wnt-3(RefSeq.: NM-030753), Wnt3a (GenBank accession No. NM-033131), Wnt-4(GenBank accession No. NM-030761), Wnt-5A (GenBank accession No. NM-003392), Wnt-5B (GenBank accession No. NM-032642), Wnt-6(GenBank accession No. NM-006522), Wnt-7A (GenBank accession No. NM-004625), Wnt-7B (GenBank accession No. NM-058238), Wnt-8A (GenBank accession No. NM-058244), Wnt-8B (GenBank accession No. NM-003393), Wnt-9A (Wnt-14) (GenBank accession No. 36 003395), Wnt-9B (Wnt-15) (Wnt-003396) (GenBank accession No. NM-OA), Wnt-1(GenBank accession No. NM-3638), Wnt-025216 (GenBank accession No. NM-3638), Wnt-11(GenBank accession NM-004626), Wnt-16(GenBank accession NM-016087)). Although each member has varying degrees of sequence identity to the family, all members encode smaller (i.e., 39-46kD), acylated, palmitoylated, secreted glycoproteins containing 23-24 conserved cysteine residues that are highly conserved at intervals (McMahon, A P et al, Trends (Trends Genet.) 1992; 8: 236-. Other native sequence Wnt polypeptides of interest include the above orthologs from any mammal, including domestic and farm animals, as well as zoo, laboratory or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, drosophila, worms, and the like.

As used herein, "Wnt pathway signaling" or "Wnt signaling" refers to the mechanism by which biologically active Wnt exerts its effect on a cell to modulate the activity of the cell. Wnt proteins modulate cellular activity by binding to Wnt receptors, which comprise proteins from the frizzled (Fzd) family of proteins, proteins from the ROR family of proteins, proteins LRP5 and LRP6 from the LRP family of proteins, the protein FRL1/crypto and the protein Derailed/Ryk. Once activated by Wnt binding, one or more Wnt receptors will activate one or more intracellular signaling cascades. These contain canonical Wnt signaling pathways; the Wnt/planar cell polarity (Wnt/PCP) pathway; wnt-calcium (Wnt/Ca)²⁺) Pathways (Giles, RH et al (2003) biochemical and biophysical letters 1653, 1-24; peifer, M.et al (1994) Development (Development) 120: 369-380; papkoff, J. et al(1996) 2128-2134 in molecular and cellular biology 16; veeman, m.t. et al (2003) developmental cells (dev.cell) 5: 367-; and other Wnt signaling pathways as are known in the art.

For example, activation of the canonical Wnt signaling pathway results in inhibition of phosphorylation of the intracellular protein β -catenin, leading to accumulation of β -catenin in the cytosol and subsequent translocation to the nucleus where β -catenin interacts with transcription factors (e.g., TCF/LEF) to activate target genes activation of the Wnt/PCP pathway activates RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control biological processes such as tissue polarity and cell motility ²⁺Causing the release of intracellular calcium ions, which activate calcium-sensitive enzymes such as Protein Kinase C (PKC), calcium-calmodulin-dependent kinase II (CamKII), or calcineurin (CaCN). The biological activity of an antibody or antigen-binding fragment thereof (e.g., Wnt-substitute) can be readily determined by assaying the activity of the above-described signaling pathway.

In certain embodiments, the functional properties of anti-LRP 5/6 antibodies and antigen-binding fragments thereof can be assessed using a variety of methods known to the skilled artisan, including, for example, affinity/binding assays (e.g., surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays) in response to Wnt, cancer cell and/or tumor growth inhibition using in vitro or in vivo models, including, but not limited to, any of the in vitro or in vivo models described herein. Other assays may test the ability of the antibodies described herein to block normal Wnt/LRP 5/6-mediated responses. The antibodies and antigen binding fragments thereof described herein can also be tested for their effect on LRP5/6 receptor internalization, in vitro and in vivo efficacy, and the like. Such assays can be performed using accepted protocols known to the skilled artisan or commercially available kits (see, e.g., modern molecular biology laboratory guidelines (Greene publication consortium, assoc, inc.) and John william father, new york); modern immunology laboratory guidelines (edited by John e.coli, Ada m.kruisbeam, David h.margulies, Ethan m.shuvach, WarrenStrober 2001, jornen publication consortium, new york).

In certain embodiments, the LRP5/6 binding antibody includes one or more of the CDRs of the antibody described herein. In this regard, it has been shown that in some cases only transfer of the VHCDR3 of an antibody can be performed while still retaining the desired specific binding (Barbas et al, Proc. Natl. Acad. Sci. USA (1995)92: 2529-2533). See also McLane et al, Proc. Natl.Acad.Sci., USA (1995)92: 5214-.

Marks et al (Bio/technology, 1992,10:779-783) describe a method of generating a repertoire of antibody variable domains in which universal primers directed at or adjacent to the 5' end of the variable domain are used to link the universal primers to the third framework region of a human VH gene to provide a repertoire of VH variable domains lacking CDR 3. Marks et al further describe how this set of libraries can be combined with the CDR3 of a particular antibody. Using similar techniques, the CDR 3-derived sequences of the presently described antibodies can be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains combined with homologous VL or VH domains to provide antibodies or antigen-binding fragments thereof that bind LRP5 and/or LRP 6. This repertoire can then be displayed in a suitable host system (e.g.the phage display system of WO 92/01047) so that suitable antibodies or antigen-binding fragments thereof can be selected. The repertoire can be comprised of at least about 10 ⁴Single member and increased by several orders of magnitude, e.g. to about 10⁶To 10⁸Or 10¹⁰Stemmer (Nature, 1994,370:389-391) also discloses similar shuffling or combinatorial techniques which describe techniques related to the β -lactamase gene, but which have observed that the methods can be used for antibody production.

A further alternative is to use random mutagenesis of one or more selected VH and/or VL genes to generate novel VH or VL regions carrying one or more CDR-derived sequences of the inventive embodiments described herein to generate mutations throughout the variable domain. This technique is described by Gram et al (1992, Proc. Natl. Acad. Sci. USA, 89: 3576-. Another method that may be used is to direct mutagenesis to the CDR regions of the VH or VL genes. Such techniques are disclosed by Barbas et al (1994, Proc. Natl. Acad. Sci. USA 91:3809-3813) and Schier et al (1996, journal of molecular biology 263: 551-.

In certain embodiments, specific VH and/or VL of the antibodies described herein can be used to screen libraries of complementary variable domains to identify antibodies with desired properties (e.g., increased affinity for LRP5 and/or LRP 6). Such methods are described, for example, in Portolano et al, J Immunol (1993)150: 880-887; clarkson et al, Nature (1991)352: 624-.

Other methods may also be used to mix and match the CDRs to identify antibodies with desired binding activity (e.g., binding to LRP5 and/or LRP 6). For example: klimka et al, J.K. Cancer (British Journal of Cancer) (2000)83:252-260 describe a screening process using mouse VL and human VH libraries in which CDR3 and FR4 are retained from the mouse VH. After obtaining the antibodies, the VH is screened against a human VL library to obtain antigen-binding antibodies. Beiboer et al, J.Mol.biol. (2000)296: 833-. After obtaining the antibodies, one VL was combined with the human VH library, in which the mouse CDR3 was retained. An antibody capable of binding to the antigen is obtained. Rader et al, Proc. Natl. Acad. Sci. USA (1998)95:8910-8915 describe a similar process to that described above for Beiboer et al.

These just described techniques are known per se in the art. However, one skilled in the art will be able to use such techniques to obtain antibodies or antigen-binding fragments thereof according to several embodiments of the invention described herein using methods routine in the art.

Also disclosed herein is a method for obtaining an antibody antigen binding domain specific for LRP5 and/or LRP6 antigens, the method comprising providing a VH domain as set forth herein, a VH domain that is an amino acid sequence variant of the VH domain, by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence; optionally combining the VH domains thus provided with one or more VL domains; and testing the VH domain or one or more VH/VL combinations to identify a specific binding member or antibody antigen binding domain specific for LRP5 and/or LRP6 and optionally having one or more desired properties. The VL domain may have an amino acid sequence substantially as shown herein. Similar methods may be employed wherein one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

In particular embodiments, the anti-LRP 5/6 antibodies and antigen binding fragments thereof are water soluble. By "water-soluble" is meant a composition that is soluble in an aqueous buffer in the absence of a detergent, typically at a concentration that provides a biologically effective dose of the polypeptide. The water-soluble composition forms a substantially homogeneous composition having a specific activity that is at least about 5% of the specific activity of the starting material from which the substantially homogeneous composition is purified, typically at least about 10%, 20%, or 30% of the specific activity of the starting material, more typically about 40%, 50%, or 60% of the specific activity of the starting material, and may be about 50%, about 90%, or greater. The anti-LRP 5/6 antibodies and antigen-binding fragments thereof (including Wnt substitutions) of the invention typically form substantially homogeneous aqueous solutions at concentrations of at least 25 μ Μ or higher, e.g., at least 25 μ Μ, 40 μ Μ or 50 μ Μ, typically at least 60 μ Μ, 70 μ Μ, 80 μ Μ or 90 μ Μ, sometimes up to 100 μ Μ, 120 μ Μ or 150 μ Μ. In other words, the compositions of the present invention generally form substantially homogeneous aqueous solutions having concentrations of about 0.1mg/ml, about 0.5mg/ml, about 1mg/ml or greater.

Epitopes that "specifically bind" or "preferentially bind" (used interchangeably herein) to an antibody or polypeptide are terms well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and/or with greater affinity than it does with an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, more readily and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to LRP5 is an antibody that binds LRP5 with greater affinity, more readily, and/or for a longer duration than it binds to LRP6 or a non-LRP 5/6 protein. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds a first target may or may not specifically or preferentially bind a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it may comprise) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

Immunological binding generally refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, due to electrostatic, ionic, hydrophilic and/or hydrophobic attractive or repulsive forces, steric forces, hydrogen bonding, van der waals forces, and other interactions. Dissociation constant (K) at interaction_d) Aspects express the strength or affinity of an immunological binding interaction, with smaller Ks_dIndicating greater affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rates of antigen binding site/antigen complex formation and dissociation, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and on geometric parameters that also affect the rate in both directions. Thus, the "association rate constant" (K) can be determined by calculating the concentration and actual rate of association and dissociation_on) And "dissociation rate constant" (K)_on)。K_off/K_onIs achieved to eliminate all parameters that are independent of affinity and are therefore equal to the dissociation constant K _d. See generally Davies et al, (1990) Annual Biochemical annals 59:439-473。

In certain embodiments, the anti-LRP 5/6 antibody is less than or equal to about 1 × 10^-4M, less than or equal to about 1 × 10^{- 5}M, less than or equal to about 1 × 10^-6M, less than or equal to about 1 × 10^-7M, less than or equal to about 1 × 10^-8M, less than or equal to about 1 × 10^-9M or at least about 1 × 10^-10K of M_DBind LRP5 and/or LRP 6. In certain embodiments, an anti-LRP 5/6 antibody described herein has a K of less than about 10,000nM, less than about 1000nM, less than about 100nM, less than about 10nM, less than about 1nM, or less than about 0.1nM_DBinding to LRP5 and/or LRP6, and in some embodiments, the antibody may have even higher affinity for one or more Fzd receptors. In certain embodiments, an anti-LRP 5/6 antibody described herein has an affinity K of about 100 picomolar, 150 picomolar, 155 picomolar, 160 picomolar, 170 picomolar, 175 picomolar, 180 picomolar, 185 picomolar, 190 picomolar, 191 picomolar, 192 picomolar, 193 picomolar, 194 picomolar, 195 picomolar, 196 picomolar, 197 picomolar, 198 picomolar, or 199 picomolar _DAnd in some embodiments, the antibody may have even higher affinity for LRP5 and/or LRP 6.

Referring to an epitope being "immunologically active" or "remaining immunologically active," the term "immunologically active" refers to the ability of an antibody (e.g., an anti-LRP 5/6 antibody) to bind to the epitope under different conditions, e.g., after the epitope has undergone reducing and denaturing conditions.

An antibody or antigen-binding fragment thereof according to certain preferred embodiments of the present application may be an antibody or antigen-binding fragment thereof that competes for binding to LRP5 and/or LRP6, or a variant of any of these antibodies or antigen-binding fragments thereof, that (i) specifically binds to an antigen and (ii) comprises a VH and/or VL domain disclosed herein, or comprises a VH CDR3 disclosed herein. Competition between antibodies can be readily determined in vitro, for example, using ELISA and/or by labeling a particular reporter molecule to one antibody that can be detected in the presence of other unlabeled antibodies to achieve identification of specific antibodies that bind the same epitope or overlapping epitopes. Accordingly, provided herein is a specific antibody or antigen-binding fragment thereof comprising a human antibody antigen-binding site that competes with an antibody described herein that binds LRP5 and/or LRP 6.

In this regard, as used herein, the terms "compete with … …," "inhibit binding," and "block binding" (e.g., refer to inhibiting/blocking Wnt binding to LRP5 and/or LRP6 or to inhibiting/blocking binding of an anti-LRP 5/6 antibody to LRP5 and/or LRP 6) are used interchangeably and encompass partial and complete inhibition/blocking. Inhibition/blocking of LRP5 and/or LRP6 by Wnt preferably reduces or alters the normal level or type of cellular signaling that occurs when Wnt binds to LRP5 and/or LRP6 without inhibition or blocking. Inhibition and blocking is also intended to encompass any measurable decrease in Wnt binding to LRP5 and/or LRP6 upon contact with the anti-LRP 5/6 antibody described herein, e.g., blocking Wnt binding to LRP5 and/or LRP6 by at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as compared to when the ligand is not contacted with the anti-LRP 5/6 antibody.

The constant regions of immunoglobulins exhibit less sequence diversity than the variable regions and are responsible for binding to many natural proteins to initiate important biochemical events. In humans, there are five different classes of antibodies, including IgA (which comprises subclasses IgA1 and IgA2), IgD, IgE, IgG (which comprises subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. The distinguishing feature between these antibody classes is their constant regions, but there can be subtle differences in the V regions.

The Fc region of an antibody interacts with a number of Fc receptors and ligands, conferring a range of important functional capabilities called effector functions. For IgG, the Fc region includes the Ig domains CH2 and CH3 and the N-terminal hinge to CH 2. An important family of Fc receptors of the IgG class is the Fc γ receptor (Fc γ R). These receptors mediate communication between antibodies and the cellular immune part of the immune system (cellular arm) (Raghavan et al, 1996, annual review in cell and developmental biology (Annu Revcell Dev Biol) 12: 181-. In humans, this family of proteins comprises: fc γ RI (CD64) comprising the isoforms Fc γ RIa, Fc γ RIb and Fc γ RIc; fc γ RII (CD32) comprising the isoforms Fc γ RIIa (comprising allotypes H131 and R131), Fc γ RIIb (comprising Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; and Fc γ RIII (CD16) comprising isoforms Fc γ RIIIa (comprising allotypes V158 and F158) and Fc γ RIIIb (comprising allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2) (Jefferis et al, 2002, handbook of immunology letters 82: 57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that may mediate some signaling event within the cell. These receptors are expressed in a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, langerhans cells, Natural Killer (NK) cells, and T cells. Formation of the Fc/fcyr complex recruits these effector cells to the site of antigen binding, thereby generally generating intracellular signaling events and important subsequent immune responses such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells. The cell-mediated reaction in which Fc γ R-expressing non-specific cytotoxic cells recognize bound antibody on target cells and subsequently cause lysis of the target cells is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al, 1996, annual review in cell and developmental biology 12: 181-220; Ghetie et al, 2000, annual review in immunology 18: 739-766; Ravetch et al, 2001, annual review in immunology 19: 275-290). The cell-mediated reaction in which Fc γ R-expressing non-specific cytotoxic cells recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells is referred to as antibody-dependent cell-mediated phagocytosis (ADCP). All Fc γ rs bind to the same region on Fc at the N-terminus of the Cg2(CH2) domain and the preceding hinge. This interaction is well characterized structurally (Sondermann et al, 2001, J. mol. biol. 309: 737. 749) and several structures of human Fc (pdb accession code 1E4K) which bind to the extracellular domain of human Fc γ RIIIb have been solved (Sondermann et al, 2000, Nature 406: 267. 273). (pdb accession codes 1IIS and 1IIX) (Radaev et al, 2001, J. Biochem. 276: 16469-16477).

The different IgG subclasses have different affinities for Fc γ R, IgG1 and IgG3 generally bind significantly better to the receptor than IgG2 and IgG4 (Jefferis et al, 2002, promiscuous immunology 82: 57-65). All Fc γ rs bind to the same region on IgG Fc, but have different affinities: high affinity binders Fc γ RI K against IgG1_dIs 10^-8M^-1Whereas the low affinity receptors Fc γ RII and Fc γ RIII are typically at 10, respectively^-6And 10^-5And (4) combining. The extracellular domains of Fc γ RIIIa and Fc γ RIIIb are 96% identical; however, Fc γ RIIIb does not have an intracellular signaling domain. Furthermore, while Fc γ RI, Fc γ RIIa/c, and Fc γ RIIIa are positive regulators of immune complex-triggered activation characterized by having an intracellular domain with an immunoreceptor tyrosine-based activation motif (ITAM), Fc γ RIIb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory. Thus, the former is referred to as the activating receptor and Fc γ RIIb is referred to as the inhibitory receptor. The receptor is expressed in different ways and at different levels on different immune cells. Another level of complexity is the presence of many Fc γ R polymorphisms in the human proteome. A particularly relevant polymorphism of clinical significance is V158/F158 Fc γ RIIIa. Human IgG1 has greater binding affinity for the V158 allotype than the F158 allotype. This difference in affinity and presumably its effect on ADCC and/or ADCP has been shown to be the anti-CD 20 antibody rituximab (rituximab) ((r))

IDEC pharmaceuticals Corporation (registered trademark of IDECpharmaceuticals Corporation) is an important determinant of efficacy. Patients with V158 allotypesGood response to rituximab treatment; however, patients with the lower affinity F158 allotype responded poorly (Cartron et al, 2002, Blood 99: 754-. Approximately 10-20% of the population are V158/V158 homozygotes, 45% of the population are V158/F158 heterozygotes, and 35-45% of the population are F158/F158 homozygotes (Lehrnbecher et al, 1999, blood, 94: 4220-. Thus, 80-90% of people are poor responders, that is, they have at least one allele of F158 Fc γ RIIIa.

The Fc region is also involved in activation of the complement cascade. In the classical complement pathway, C1 binds with its C1q subunit to the Fc fragment of IgG or IgM that has formed a complex with one or more antigens. In certain embodiments of the invention, modifications to the Fc region include modifications that alter (enhance or reduce) the ability of the x-specific antibodies described herein to activate the complement system (see, e.g., U.S. Pat. No. 7,740,847). To assess complement activation, Complement Dependent Cytotoxicity (CDC) assays can be performed (see, e.g., Gazzano-Santoro et al, journal of immunological methods, 202:163 (1996)).

Thus, in certain embodiments, the invention provides anti-LRP 5/6 antibodies with modified Fc regions that have altered functional properties, such as reduced or enhanced CDC, ADCC, or ADCP activity or enhanced binding affinity to a particular fcyr or increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in published U.S. patents 7,317,091; 7,657,380, respectively; 7,662,925, respectively; 6,538,124, respectively; 6,528,624, respectively; 7,297,775, respectively; 7,364,731, respectively; published U.S. applications US 2009092599; US 20080131435; US 20080138344; and published international application WO 2006/105338; WO 2004/063351; WO 2006/088494; WO 2007/024249.

Thus, in certain embodiments, an antibody variable domain having the desired binding specificity is fused to an immunoglobulin constant domain sequence. In certain embodiments, the fusion is with a linker comprising a hinge region, C _H2 and C_HThe Ig heavy chain constant domain of at least part of region 3 is fused. It is preferred to have a light chain linkage present in at least one of the fusions comprisingThe first heavy chain constant region (C) at the required site_H1). The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in the examples, as unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. However, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector when expression of at least two polypeptide chains at equal ratios results in high yield or when the ratios have no significant effect on the yield of the desired chain combination.

The antibodies of the invention (and antigen-binding fragments and variants thereof) may also be modified to include epitope tags or labels, for example, for purification or diagnostic applications. There are many linking groups known in the art for preparing antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP 0425235B 1 and Chari et al, cancer research 52: 127-. As disclosed in the above patents, the linking group comprises a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group or an esterase labile group, preferably a disulfide group and a thioether group.

In another contemplated embodiment, an LRP5/6 specific antibody described herein can be conjugated or operably linked to another therapeutic compound referred to herein as a conjugate. The conjugate may be a cytotoxic agent, chemotherapeutic agent, cytokine, anti-angiogenic agent, tyrosine kinase inhibitor, toxin, radioisotope, or other therapeutically active agent. Chemotherapeutic agents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors and other therapeutic agents have been described above, and all of these above-mentioned therapeutic agents can be used as antibody conjugates.

Bifunctional derivatives such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Iminotetrahydrothiophene (IT), imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), bifunctional protein coupling agents such as aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexamethylenediamine), bis-diazo derivatives (e.g., bis- (p-diazobenzoyl) -ethylenediamine), diisocyanates (e.g., 2, 6-tolylene diisocyanate), and bis-active fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene) are used to prepare immunoconjugates. Specific coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al, J. biochem., 173:723-737[1978]) and N-succinimidyl-4- (2-pyridylthio) valerate (SPP) to provide disulfide bonds. The linker may be a "cleavable linker" that facilitates the release of one or more cleavable components. For example, acid-labile linkers can be used (cancer research 52:127- & 131 (1992); U.S. Pat. No. 5,208,020).

In certain embodiments, the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a monoclonal antibody. In certain embodiments, it is humanized.

In certain embodiments, the invention further provides isolated nucleic acids encoding an antibody or antigen-binding fragment thereof described herein, e.g., a nucleic acid encoding a CDR or VH or VL domain described herein. Nucleic acids include DNA and RNA. These and related embodiments may comprise polynucleotides encoding antibodies that bind LRP5 and/or LRP6 described herein. The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, from which the isolated polynucleotide (1) is not associated with all or a portion of the polynucleotide, (2) is linked to a polynucleotide to which it is not naturally linked, or (3) does not occur in nature as part of a larger sequence.

The term "operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent function under appropriate conditions. For example, a transcriptional control sequence "operably linked" to a protein coding sequence is ligated thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

The term "control sequence" as used herein refers to a polynucleotide sequence that can affect the expression, processing, or intracellular localization of a coding sequence to which it is linked or operably linked. The nature of such control sequences may depend on the host organism. In particular embodiments, the transcriptional control sequences of prokaryotes may comprise a promoter, a ribosome binding site, and a transcriptional termination sequence. In other particular embodiments, the eukaryotic transcription control sequence may comprise a promoter that includes one or more recognition sites for a transcription factor, a transcription enhancer sequence, a transcription termination sequence, and a polyadenylation sequence. In certain embodiments, a "control sequence" can comprise a leader sequence and/or a fusion partner sequence.

The term "polynucleotide" as referred to herein means a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of either nucleotide type. The modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3' -dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothionate, phosphoroanillylate, and phosphoramidate. The term "polynucleotide" specifically encompasses single-stranded and double-stranded forms of DNA.

The term "naturally occurring nucleotide" encompasses deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" encompasses nucleotides having modified or substituted sugar groups and the like.

The term "oligonucleotide linkage" encompasses oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphorthioaniline, phosphoranilate, phosphoroamidate, and the like. See, e.g., LaPlanche et al, 1986, nucleic acids research, 14: 9081; stec et al, 1984, J.Am.Chem.Soc.106: 6077; stein et al, 1988, nucleic acids research 16: 3209; zon et al, 1991, "Anti-Cancer drug design (Anti-Cancer drug design)," 6: 539; zon et al, 1991, "oligonucleotides and analogs: practical methods (OLIGONUCLEOTIDES ANDANALOGUES: APRACTICAL APPROACH), pp 87 to 108 (edited by F. Eckstein), Oxford University Press, Oxford England, Oxford, Ouk; stec et al, U.S. patent No. 5,151,510; uhlmann and Peyman,1990, "Chemical Reviews (Chemical Reviews)," 90:543, the disclosure of which is hereby incorporated by reference for any purpose. The oligonucleotide may comprise a detectable label to enable detection of the oligonucleotide or its hybridization.

The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or controls the expression of an inserted heterologous nucleic acid sequence. If introns are present, expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing.

As will be understood by those skilled in the art, polynucleotides may comprise genomic sequences, extra-genomic and plasmid-encoded sequences, and smaller engineered gene segments that express or may be suitable for expression of proteins, polypeptides, peptides, and the like. Such segments may be naturally isolated or synthetically modified by those skilled in the art.

As will also be appreciated by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules can include HnRNA molecules that contain introns and correspond in a one-to-one manner to DNA molecules, as well as mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide according to the present disclosure, and the polynucleotide may, but need not, be linked to other molecules and/or support materials. The polynucleotide may comprise the native sequence or may comprise a sequence encoding a variant or derivative of this sequence.

Thus, according to these and related embodiments, the disclosure also provides polynucleotides encoding the anti-LRP 5/6 antibodies and antigen-binding fragments thereof described herein. In certain embodiments, polynucleotides are provided that include some or all of the polynucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein, as well as the complement of such polynucleotides.

It will be appreciated by those of ordinary skill in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the antibodies described herein. Some of these polynucleotides have minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence encoding the antibody that binds LRP5 and/or LRP 6. However, the present disclosure expressly contemplates polynucleotides that vary due to differences in codon usage. In certain embodiments, sequences that have been codon optimized for mammalian expression are specifically contemplated.

Thus, in another embodiment of the invention, mutagenesis methods such as site-specific mutagenesis may be used to prepare variants and/or derivatives of the antibodies described herein. In this way, specific modifications of the polypeptide sequence can be made by mutagenesis of the underlying polynucleotide encoding it. These techniques provide a direct method for making and testing sequence variants (e.g., including one or more of the considerations described above) by introducing one or more nucleotide sequence changes into a polynucleotide.

Site-specific mutagenesis allows the generation of mutants by: specific oligonucleotide sequences encoding the DNA sequences desired to be mutated and a sufficient number of adjacent nucleotides are used to provide primer sequences of sufficient size and sequence complexity to form a stable duplex on both sides of the traversed deletion junction. Mutations can be employed in selected polynucleotide sequences to improve, alter, reduce, modify or otherwise alter the properties of the polynucleotide itself and/or to alter the properties, activity, composition, stability or primary sequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate mutagenizing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein to alter one or more properties of the encoded polypeptide, such as the binding affinity of the antibody or antigen-binding fragment thereof or the function of a particular Fc region or the affinity of the Fc region for a particular fcyr. Site-specific mutagenesis techniques are well known in the art and are widely used to generate variants of polypeptides and polynucleotides. For example, site-specific mutagenesis is commonly used to alter specific portions of a DNA molecule. In such embodiments, primers are employed that typically include about 14 to about 25 nucleotides or so in length, with about 5 to about 10 residues being altered on both sides of the junction of the sequences.

As will be appreciated by those skilled in the art, site-specific mutagenesis techniques typically employ phage vectors that exist in both single-stranded and double-stranded form. Typical vectors that can be used for site-directed mutagenesis include vectors such as M13 phage. These bacteriophages are readily commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also commonly used for site-directed mutagenesis which eliminates the step of transferring the gene of interest from the plasmid to the phage.

In many embodiments, the nucleic acid encoding the subject monoclonal antibody is introduced directly into a host cell species, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. Antibodies of the present disclosure are prepared using standard techniques well known to those skilled in the art and in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequence may be used to determine the appropriate nucleic acid sequence encoding a particular antibody disclosed herein. The nucleic acid sequence may be optimized to reflect the specific codon "bias" of various expression systems according to standard methods well known to those skilled in the art.

According to certain related embodiments, there is provided a recombinant host cell comprising one or more of the constructs described herein; nucleic acid encoding any antibody, CDR, VH or VL domain or antigen binding fragment thereof; and methods of producing the encoded products, the methods comprising expression from an encoding nucleic acid directed thereto. Expression can be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the antibody or antigen-binding fragment thereof can be isolated and/or purified using any suitable technique, and then used as desired.

The antibodies or antigen-binding fragments thereof as provided herein, as well as the encoding nucleic acid molecules and vectors, can be isolated and/or purified, e.g., from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free of the original nucleic acid or gene other than the sequence encoding the polypeptide having the desired function. Nucleic acids may include DNA or RNA, and may be wholly or partially synthetic. Unless the context requires otherwise, reference to a nucleotide sequence set forth herein encompasses a DNA molecule having the specified sequence, and encompasses an RNA molecule having the specified sequence with U substituted for T.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and the like. A common preferred bacterial host is E.coli.

Expression of antibodies and antigen-binding fragments in prokaryotic cells such as E.coli is well established in the art. For a review see, for example, Pluckthun, A. & Bio/technology 9:545-551 (1991). As an alternative to the production of antibodies or antigen-binding fragments thereof, expression in eukaryotic cells in culture may also be obtained by the skilled person, see for example recent reviews, e.g. Ref, M.E (1993) current opinion on biotechnology (curr. opinion Biotech.) 4: 573-576; trill J.J. et al (1995) Current opinion on Biotechnology 6: 553-560.

Suitable vectors may be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences. The vector may be a plasmid, a virus, such as a phage or a phagemid, as desired. For additional details, see, e.g., "molecular cloning: laboratory manuals ": 2 nd edition, Sambrook et al, 1989, Cold Spring Harbor laboratory Press (Cold Spring Harbor laboratory Press). A number of known techniques and protocols for manipulating nucleic acids, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in the modern molecular biology laboratory Manual, second edition, edited by Ausubel et al, John Willi's parent subsidiary, 1992, or later updates.

The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the antibodies described herein has been introduced or is capable of being introduced, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any of the antibodies described herein. The term encompasses progeny of a parent cell, whether or not the progeny is morphologically or genetically identical to the original parent, so long as the selected gene is present. Thus, methods comprising introducing such nucleic acids into host cells are also contemplated. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia or viruses directed against insect cells, baculovirus). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using phage. After introduction, expression from the nucleic acid may be caused or allowed, for example, by culturing the host cell under conditions in which the gene is expressed. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration may be facilitated by inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

In certain embodiments, the invention also provides a method comprising using a construct as described above in an expression system to express a particular polypeptide, such as an LRP5 or LRP6 specific antibody described herein. The term "transduction" is used to refer to the transfer of a gene from one bacterium to another, usually by phage. "transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and when exogenous DNA has been introduced inside the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology (Virology) 52: 456; sambrook et al, 2001, MOLECULAR CLONING, A LABORATORY Manual (Molecula CLONING, A Labratory Manual), Cold spring harbor LABORATORY; davis et al, 1986, BASIC METHODS of MOLECULAR BIOLOGY (BASIC METHODS IN MOLECULAR BIOLOGY), Emesweil (Elsevier); and Chu et al, 1981, Gene (Gene) 13: 197. Such techniques may be used to introduce one or more exogenous DNA moieties into a suitable host cell.

The term "transformation" as used herein refers to an alteration of the genetic properties of a cell, and when a cell has been modified to contain new DNA, the cell has been transformed. For example, in the case of a cell that is genetically modified from its native state, the cell is transformed. Following transfection or transduction, the transforming DNA may recombine with the DNA of the cell by physically integrating into the chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. When DNA replicates as a cell divides, the cell is considered to have been stably transformed. The term "naturally-occurring" or "native" when used in conjunction with biological materials, such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials that are found in nature and not manipulated by humans. Similarly, "non-naturally occurring" or "non-natural" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by humans.

The terms "polypeptide", "protein" and "peptide" and "glycoprotein" are used interchangeably and are meant to refer to a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein may comprise a plurality of chains non-covalently and/or covalently linked by peptide bonds and having the sequence of a native protein, i.e. a protein produced by a naturally occurring and in particular non-recombinant cell, or genetically engineered or recombinant cell, and comprising a molecule having the amino acid sequence of a native protein, or a molecule having the deletion, addition and/or substitution of one or more amino acids of a native sequence. The terms "polypeptide" and "protein" specifically encompass antibodies that bind to the LRP and/or LRP6 receptors of the present disclosure, or sequences having a deletion, addition and/or substitution of one or more amino acids of an anti-LRP 5 antibody or an anti-LRP 6 antibody. Thus, a "polypeptide" or "protein" may comprise one (referred to as a "monomer") or a plurality (referred to as a "multimer") of amino acid chains.

The term "isolated protein" as referred to herein means the subject protein: (1) free of at least some other proteins with which the subject protein will typically be found in nature; (2) substantially free of other proteins from the same source, e.g., from the same species; (3) expressed by cells from different species; (4) has been separated from at least about 50% of the polynucleotide, lipid, carbohydrate, or other material with which it is naturally associated; (5) not associated (by covalent or non-covalent interactions) with the protein moiety with which the "isolated protein" is naturally associated; (6) and polypeptides not naturally associated therewith (by covalent or non-covalent interactions); or (7) does not occur in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants that would be found in its natural environment and would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

One or more amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. For example, amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes into a polynucleotide encoding the antibody or its chain or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final antibody, provided that the final construct has the desired properties (e.g., high affinity for binding to LRP5 and/or LRP 6). Amino acid changes can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the changes and modifications described above for the polypeptides of the invention may be included in the antibodies of the invention.

The present disclosure provides variants of the antibodies disclosed herein. In certain embodiments, the binding of such variant antibodies or antigen binding fragments, or CDRs thereof, to LRP5 and/or LRP6 is at least about 50%, at least about 70%, and in certain embodiments at least about 90% relative to the binding of the antibody sequences specifically set forth herein to LRP5 and/or LRP 6. In further embodiments, such variant antibodies or antigen-binding fragments or CDRs thereof bind LRP5 and/or LRP6 with greater affinity than the antibodies set forth herein, e.g., binding quantitatively is at least about 105%, 106%, 107%, 108%, 109%, or 110% relative to binding of the antibody sequences specifically set forth herein.

In particular embodiments, an antibody or antigen-binding fragment thereof (e.g., Fab, scFv, VHH or sdAb, or Wnt substitute) can comprise: a) a heavy chain variable region comprising: i. a CDR1 region having an amino acid sequence identical to a heavy chain CDR1 region of a selected antibody described herein; a CDR2 region having an amino acid sequence identical to a heavy chain CDR2 region of the selected antibody; a CDR3 region having an amino acid sequence identical to a heavy chain CDR3 region of the selected antibody; and/or b) a light chain variable domain comprising: i. a CDR1 region having an amino acid sequence identical to a light chain CDR1 region of the selected antibody; a CDR2 region having an amino acid sequence identical to a light chain CDR2 region of the selected antibody; a CDR3 region having an amino acid sequence identical to a light chain CDR3 region of the selected antibody; wherein the antibody specifically binds to the selected target (e.g., LRP5 and/or LRP 6). In further embodiments, the antibody or antigen-binding fragment thereof is a variant antibody, wherein the variant comprises the same heavy and light chains as the selected antibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8 amino acid substitutions in the CDR regions of the selected antibody, or in certain embodiments, there may be 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. Substitutions may be in the CDRs in the VH and/or VL regions. (see, e.g., Muller,1998 Structure 6:1153 & 1167).

In particular embodiments, the antibody or antigen-binding fragment thereof (e.g., Fab, scFv, VHH or sdAb, or Wnt substitute) may comprise one or more, two or more, three or more, four or more, five or more, or six of the CDRs identified in table 1A for any particular antibody. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: CDRH1 comprising or consisting of any one of SEQ ID NOs 34-172; CDRH2 comprising or consisting of any one of SEQ ID NO 173-312; CDRH3 comprising or consisting of any one of SEQ ID NO 313-485; CDRL1 comprising or consisting of any of SEQ ID NO 486-524; CDRL2 comprising or consisting of any one of SEQ ID NO: 525-556; and/or CDRL3 comprising or consisting of any of SEQ ID NO: 557-607.

In particular embodiments, a subject antibody (e.g., Fab, scFv, VHH, or sdAb, or Wnt substitute) can have: a) a heavy chain variable region having an amino acid sequence at least 80% identical, at least 95% identical, at least 90% identical, at least 95% identical, or at least 98% identical or 99% identical to the heavy chain variable region of an anti-LRP 5/6 antibody described herein; and/or b) a light chain variable region having an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical or 99% identical to a light chain variable region of an anti-LRP 5/6 antibody described herein. The amino acid sequences of illustrative heavy and/or light chain regions are shown in SEQ ID NOS: 1-24.

Representative polypeptides (e.g., variant LR provided herein)P%/6 specific antibodies, e.g., antibody proteins having antigen binding fragments provided herein) can be determined by conventional methods such that, for example, substitution, addition, deletion, or insertion of one or more amino acids with selected natural or unnatural amino acids can be virtually modeled for the purpose of determining whether the thus-derived structural variants retain the space-filling properties of the presently disclosed species. See, e.g., Donate et al, 1994, "protein science (prot. Sci.) 3: 2378; bradley et al, science 309:1868-1871 (2005); Schueler-Furman et al, science 310:638 (2005); dietz et al, Proc. Natl. Acad. Sci. USA 103:1244 (2006); dodson et al, Nature 450:176 (2007); qian et al, Nature 450:259 (2007); raman et al, science 327: 1014-. Some further non-limiting examples of computer algorithms that may be used in these and related embodiments (as for rational design of its LRP5/6 specific antibody antigen binding domain as provided herein) include VMDs, which are molecular visualization programs for displaying, animating and analyzing large biomolecule systems using 3-D graphs and built-in scripts, (see website ks, uiuc, edu/Research/VMD of the theoretical and Computational Biophysics Group of University of ibandron-Champagne, the University of Illinois, and urbanana-Champagne; many other computer programs are known in the art and available to the skilled artisan and allow determination of atomic dimensions from space-filling models (van der waals radii) of energy-minimized conformations; GRIDs, which attempt to determine high affinity to different chemical groups, thereby enhancing the bonded area; a Monte Carlo (Monte Carlo) search, which computes a mathematical alignment; and CHARMM (Brooks et al (1983) J.C.chem.). 4:187- & 217) and AMBER (Weiner et al (1981) J.C.chem. & 106:765) for evaluation of force field calculations and analyses (see also Eisenfield et al (1991) J.Physiol.). 261: C376-386; Lybrand (1991) J.Pharmacology. & gt 46: 49-54; Fromwitz. 1990) Biotechnology (Biotechniques) & 8:640- & 644; Burbam et al (1990) Proteins (Proteins) 7: 99-111; Pedersen (1985) environmental health prospects 61: 185-190; and Kini et al (1991) journal of biomolecular Structure and kinetics (J.Biomol. Structure. Dyn.) 9: 475-. Various suitable computer programs are also commercially available, for example

(Munich, Germany).

In particular embodiments, the disclosure provides antibodies or antigen-binding fragments thereof that bind to the E3E4 region of LRP 6. in particular embodiments, it binds to the E3 β -propeller region of LRP 6. in certain embodiments, it binds to a region of LRP6 that includes or consists of amino acid residues 637-878, where the amino acid sequence and numbering is consistent with that described in the examples

) Comprises the following steps: arg639, Ala640, Lys622, Glu663, Ile681, Ser682, Lys684, Asp705, Tyr706, Glu708, Thr724, Gly725, Arg751, Try767, Gly768, Gly769, Arg792, Leu810, Asp811, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875, and Met877 of LRP 6. In another embodiment, the interatomic distance between the core interaction site (Lrp6E3E4 and VHH36 is less than or equal to

) Comprises the following steps: glu663, Ser665, Ile681, Tyr706, Glu708, Thr724, Ser749, Arg751, Trp767, Gly768, Arg792, Leu810, Asn813, Pro833, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Try875, and Met877 of LRP 6.

In another embodiment of the invention, anti-LRP 5/6 antibodies and humanized forms thereof are derivedSelf-rabbit monoclonal antibodies, and in particular uses

And (4) technical generation. These antibodies are advantageous because they require minimal sequence modification to help retain functional properties after humanization using mutant lineage-guided (MLG) humanization techniques (see, e.g., U.S. patent No. 7,462,697). Thus, illustrative methods for making anti-antibodies of the disclosure include, for example, those described in U.S. patents 5,675,063 and 7,429,487

Rabbit monoclonal antibody technology. In this regard, in certain embodiments, the anti-LRP 5/6 antibodies of the disclosure are produced in rabbits. In particular embodiments, rabbit-derived immortalized B lymphocytes capable of fusing with rabbit spleen cells are used to generate hybrid cells that produce antibodies. Immortalized B lymphocytes do not express endogenous immunoglobulin heavy chains in a detectable manner and may, in certain embodiments, contain altered immunoglobulin heavy chain encoding genes.

Composition comprising a metal oxide and a metal oxide

Also disclosed are pharmaceutical compositions comprising an anti-LRP 5/6 antibody or antigen-binding fragment thereof described herein (e.g., a Wnt substitute) and one or more pharmaceutically acceptable diluents, carriers, or excipients. In particular embodiments, the pharmaceutical composition further comprises one or more Wnt polypeptides or Norrin polypeptides.

In additional embodiments, pharmaceutical compositions are also disclosed that include a polynucleotide that includes a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof described herein (e.g., a Wnt substitute), and one or more pharmaceutically acceptable diluents, carriers, or excipients. In particular embodiments, the pharmaceutical composition further comprises one or more polynucleotides comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a 5 'cap sequence and/or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same polynucleotide.

In additional embodiments, pharmaceutical compositions are also disclosed that include an expression vector (e.g., a viral vector) that includes a polynucleotide that includes a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof described herein (e.g., a Wnt substitute), and one or more pharmaceutically acceptable diluents, carriers, or excipients. In particular embodiments, the pharmaceutical composition further includes an expression vector (e.g., a viral vector) that includes a polynucleotide that includes a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same polynucleotide (e.g., expression cassette).

The invention further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding an anti-LRP 5/6 antibody and antigen-binding fragments thereof described herein and one or more pharmaceutically acceptable diluents, carriers or excipients. In particular embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same polynucleotide (e.g., expression cassette) and/or in the same cell. In particular embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In particular embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

The present disclosure contemplates pharmaceutical compositions comprising a first molecule for delivering an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) as a first active agent and a second molecule for delivering a Wnt polypeptide or a Norrin polypeptide. The first and second molecules may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first molecule and the second molecule may each be independently selected from the following types of molecules: a polypeptide, a small organic molecule, a nucleic acid encoding a first or second agent (optionally, DNA or mRNA, optionally, modified RNA), a vector comprising a nucleic acid sequence encoding a first or second agent (optionally, an expression vector or a viral vector), and a cell comprising a nucleic acid sequence encoding a first or second agent (optionally, an expression cassette).

The subject molecules, alone or in combination, can be combined with pharmaceutically acceptable carriers, diluents, excipients and agents useful in preparing generally safe, non-toxic and desirable formulations, and including excipients acceptable for use in mammals such as humans or primates. Such excipients may be solid, liquid, semi-solid, or gaseous in the case of an aerosol composition. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, ringer's solution, dextrose solution and 5% human serum albumin. Supplementary active compounds may also be incorporated into the formulation. The solution or suspension for the formulation may comprise: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents, such as tween 20 for preventing polymerization; and compounds for regulating osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with an acid such as hydrochloric acid or sodium hydroxide or a base. In particular embodiments, the pharmaceutical composition is sterile.

The pharmaceutical compositions may further comprise sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or Phosphate Buffered Saline (PBS). In some cases, the compositions are sterile and should be fluid to the extent that easy injection is possible. In certain embodiments, the compositions are stable under the conditions of manufacture and storage, and are preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption (e.g., aluminum monostearate and gelatin).

Sterile solutions can be prepared by incorporating the required amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof (or encoding polynucleotide or cells comprising the same) and one or a combination of the above-listed ingredients in an appropriate solvent, followed by filter sterilization, as desired. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the antibody or antigen-binding fragment thereof from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These liposome suspensions can be prepared according to methods known to those skilled in the art.

It may be advantageous to formulate the pharmaceutical composition in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active antibody or antigen-binding fragment thereof calculated to bind the desired pharmaceutical carrier to produce the desired therapeutic effect. The specification for a dosage unit form is dictated by and directly dependent on the unique characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art in compounding such active antibodies or antigen-binding fragments thereof for use in treating an individual.

The pharmaceutical composition may be contained in a container, package, or dispenser (e.g., a syringe, such as a pre-filled syringe) with instructions for administration.

The pharmaceutical compositions of the invention encompass any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other compound capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof when administered to an animal, including a human.

The invention encompasses pharmaceutically acceptable salts of the anti-LRP 5/6 antibodies or antigen-binding fragments thereof (e.g., Wnt substitutes) described herein. The term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects thereto. Various pharmaceutically acceptable salts are known in the art and are described, for example, in the following: remington's Pharmaceutical Sciences, 17 th edition, Alfonso R.Gennaro (eds.), Mark Publishing Company, Easton, Pa., USA (Mark Publishing Company, Easton, Pa., USA),1985 (and its more recent versions); encyclopedia of Pharmaceutical Technology (encyclopedia of Pharmaceutical Technology), 3 rd edition, james swartrick (eds.), lngfmann health care, Inc. of new york, USA (information Healthcare USA, NY, USA), 2007; and journal of pharmaceutical science (J.pharm.Sci.) 66:2 (1977). Furthermore, for a review of suitable salts, see Stahl and Wermuth, handbook of pharmaceutically acceptable salts: properties, Selection, and Use (Handbook of pharmaceutical Salts, Selection, and Use) "(Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metals or amines such as alkali metals and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al, "Pharmaceutical Salts", the journal of Pharmaceutical sciences (j. pharma Sci), 1977,66, 119). Base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ slightly from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention, the salts are equivalent to their respective free acids.

In some embodiments, the pharmaceutical compositions provided herein include a therapeutically effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof described herein (e.g., a Wnt-substitute) in admixture with pharmaceutically acceptable carriers, diluents, and/or excipients, such as saline, phosphate buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars and the like are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, ringer's and Hank's (Hank's) solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, the formulation is stable for at least six months at 4 ℃.

In some embodiments, the pharmaceutical compositions provided herein include buffers such as Phosphate Buffered Saline (PBS) or sodium phosphate/sulfate, tris buffers, glycine buffers, sterile water, and other buffers known to those of ordinary skill, such as those described by Good et al (1966) biochemistry 5: 467. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

Application method

The present disclosure also provides methods for using LRP5/6 specific antibodies, antigen binding fragments thereof (e.g., Wnt substitutes), disclosed herein, for example, to modulate the Wnt signaling pathway (e.g., increase or decrease Wnt signaling), as well as methods for administering Fzd-specific antibodies, antigen binding fragments thereof, and Wnt substitutes disclosed herein in various therapeutic settings. Provided herein are therapeutic methods of using antibodies or antigen-binding fragments thereof that bind to one or more Fzd receptors. In one embodiment, an antibody or antigen-binding fragment thereof of the invention is provided to a subject having a disease involving inappropriate or deregulated Wnt signaling (e.g., increased or decreased Wnt signaling).

Increasing Wnt pathway signaling and related therapeutic methods

In certain embodiments, an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) can be used to increase Wnt signaling in a tissue or cell. Thus, in some aspects, the invention provides a method for increasing Wnt signaling in a tissue or cell or enhancing Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) disclosed herein, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway agonist. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In particular embodiments, the cell is a cultured cell and the contacting occurs in vitro. In certain embodiments, the method comprises further contacting the tissue or cell with one or more Wnt polypeptides or Norrin polypeptides.

In a related aspect, the invention provides a method for increasing Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) of the invention. In certain embodiments, the target tissue or target cell is also contacted with a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a 5 'cap sequence and/or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same polynucleotide.

In a related aspect, the invention provides a method for increasing Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute). In certain embodiments, the tissue or cell is also contacted with a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may include a promoter sequence operably linked to a nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same vector (e.g., the same expression cassette).

In a related aspect, the invention provides a method for increasing Wnt signaling in a tissue, the method comprising contacting the tissue with an effective amount of a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) of the invention. In certain embodiments, the tissue is also contacted with a cell comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same cell. In particular embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In certain embodiments, the cell is transduced with a vector comprising an expression cassette encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) or a Wnt polypeptide or a Norrin polypeptide. In particular embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

anti-LRP 5/6 antibodies and antigen-binding fragments thereof (e.g., Wnt substitutes) can be used to treat diseases, disorders, or conditions, for example, by increasing Wnt signaling in a target cell, tissue, or organ. Thus, in some aspects, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or for which increasing Wnt signaling would provide a therapeutic benefit) in a subject in need thereof, comprising contacting the subject with an effective amount of a composition of the disclosure. In particular embodiments, the composition is a pharmaceutical composition comprising any one of the following: an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute); a polynucleotide comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute), e.g., a DNA or mRNA, optionally a modified mRNA; a vector, e.g., an expression vector or a viral vector, comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute); a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute), e.g., a cell transduced with an expression vector or viral vector encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute). In particular embodiments, the disease or condition is a pathological disease or disorder or an injury, such as an injury caused by a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition includes damaged tissue repair, healing, or regeneration, or may benefit from increased tissue repair, healing, or regeneration. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to the subject.

In certain embodiments, the method comprises further contacting the subject with a pharmaceutical composition comprising one or more Wnt polypeptides or Norrin polypeptides. The present disclosure contemplates contacting a subject with a first molecule for delivering an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) as a first active agent and a second molecule for delivering a Wnt polypeptide or a Norrin polypeptide. The first and second molecules may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first molecule and the second molecule may each be independently selected from the following types of molecules: a polypeptide, a small organic molecule, a nucleic acid encoding a first or second agent (optionally, DNA or mRNA, optionally, modified RNA), a vector comprising a nucleic acid sequence encoding a first or second agent (optionally, an expression vector or a viral vector), and a cell comprising a nucleic acid sequence encoding a first or second agent (optionally, an expression cassette).

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) disclosed herein. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a 5 'cap sequence and/or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same polynucleotide.

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute). In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may include a promoter sequence operably linked to a nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same vector (e.g., the same expression cassette).

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute). In certain embodiments, the subject is also contacted with a cell comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) and the nucleic acid sequence encoding the Wnt polypeptide or the Norrin polypeptide are present in the same cell. In particular embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In certain embodiments, the cell is transduced with a vector comprising an expression cassette encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) or a Wnt polypeptide or a Norrin polypeptide. In particular embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

Wnt signaling plays a key role in the development and maintenance of stem cells. Reactivation of Wnt signaling is associated with regeneration and repair of most tissues following injury and disease. It is expected that anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) molecules will provide healing and tissue repair benefits in response to injury and disease. Causes of tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infection and inflammation, traumatic injury, toxin/metabolism-induced toxicity, or other pathological conditions. Wnt signaling and enhancers of Wnt signaling have been shown to activate adult tissue resident stem cells. In some embodiments, the compounds of the invention are administered for the treatment of diseased or damaged tissue, for tissue regeneration, and for cell growth and proliferation and/or for tissue engineering.

For example, the compositions of the present invention may be used to promote or enhance bone growth or regeneration, bone grafting, fracture healing, treatment of osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injury (including vertebral compression fractures), preoperative spinal surgical optimization, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implantation, periodontal disease, maxillofacial reconstruction, and jaw necrosis. It can also be used for treating alopecia; enhancing regeneration of sensory organs (e.g., treating hearing loss, including regeneration of internal and external auditory hair cells), treating vestibular hypofunction, treating macular degeneration, treating retinopathies (including vitreoretinopathy, diabetic retinopathy, other retinal degenerative diseases), fukes' dystrophy, other corneal diseases, and the like; treating stroke, traumatic brain injury, alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy due to sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood-brain barrier. The compositions of the invention may also be used for the treatment of oral mucositis, the treatment of short bowel syndrome, Inflammatory Bowel Disease (IBD) (including Crohn's Disease (CD) and Ulcerative Colitis (UC), in particular CD with fistula formation), other gastrointestinal disorders; treating metabolic syndrome, dyslipidemia, treating diabetes, treating pancreatitis, conditions in which exocrine or endocrine pancreatic tissue is damaged; conditions in which enhanced epidermal regeneration is desired (e.g., epidermal wound healing), treatment of diabetic foot ulcers, syndromes involving underdeveloped teeth, nails, or skin, etc., conditions in which angiogenesis is beneficial; treating myocardial infarction, coronary artery disease, heart failure; enhancing growth of hematopoietic cells (e.g., enhancing hematopoietic stem cell transplantation from bone marrow), mobilizing peripheral blood, treating immunodeficiency, graft-versus-host disease, and the like; treating acute kidney injury and chronic kidney disease; treating lung diseases, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis (including idiopathic pulmonary fibrosis), enhancing regeneration of lung tissue. The compositions of the invention may also be used to enhance hepatocyte regeneration, for example liver regeneration, to treat cirrhosis, to enhance liver transplantation, to treat acute liver failure, to treat chronic liver disease undergoing hepatitis c or hepatitis b virus infection or post-viral drug therapy, alcoholic liver disease, alcoholic hepatitis, non-alcoholic liver disease with steatosis or steatohepatitis, and the like. The compositions of the invention can treat diseases and disorders, including but not limited to conditions in which regenerative cell growth is desired.

Human genetics involving loss-of-function or gain-of-function mutations in Wnt signaling components show strong evidence supporting potentiation of Wnt signaling for bone growth. Conditions in which enhanced bone growth is desired may include, but are not limited to, bone fractures, implants, ingrowth around prosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, vertebral compression fractures, preoperative optimization of spinal surgery, jaw necrosis, dental implants, periodontal disease, maxillofacial reconstruction, and the like. An anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) potentiates and promotes Wnt signaling critical to promoting bone regeneration. Methods for regenerating bone tissue benefit from administration of the compounds of the invention, which may be systemic or localized. In some embodiments, bone marrow cells are exposed to a molecule of the invention, thereby activating stem cells within the bone marrow.

In some embodiments, bone regeneration is enhanced by contacting a responsive cell population (e.g., bone marrow, osteoprogenitor cells, stem cells, etc.) with an effective dose of an anti-LRP 5/6 antibody or antigen-binding fragment thereof disclosed herein (e.g., a Wnt substitute). Methods for bone tissue regeneration benefit from administration of an anti-LRP 5/6 antibody or antigen binding fragment thereof (e.g., Wnt substitute) disclosed herein, which may be systemic or localized. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, for example by loading onto an optionally biodegradable matrix, and optionally provide sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like.

Compositions comprising one or more anti-LRP 5/6 antibodies or antigen-binding fragments thereof (e.g., Wnt substitutes) disclosed herein can be used to treat skeletal tissue defects in vivo. By "bone tissue defect" is meant a defect of bone or other bone connective tissue at any site where restoration of bone or connective tissue is desired, regardless of how the defect is caused, for example, regardless of the result of surgical intervention, tumor resection, ulceration, implantation, fracture, or other traumatic or degenerative conditions. The composition of the invention may be used as part of a protocol for restoring cartilage function to connective tissue, for repairing defects or damage to cartilage tissue such as degenerative wear and arthritis, tissue trauma, meniscal tear displacement, meniscectomy, joint dislocation caused by ligament tears, joint misalignment, bone fractures or genetic diseases.

anti-LRP 5/6 antibodies or antigen-binding fragments thereof (e.g., Wnt substitutes) may also be used to treat periodontal disease. Periodontal disease is a major cause of tooth loss and is associated with a variety of systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contact with a responsive cell population. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, followed by implantation of activated stem or progenitor cells. The molecule may be localized to the site of action, for example by loading onto an optionally biodegradable matrix, and optionally provide sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.

Studies have shown that Wnt signaling and the biology of R-vertebrates can promote the regeneration of sensory hair cells in the inner ear after injury, aging or degeneration. Loss of sensory hair cells in the inner ear, which is involved in hearing loss or vestibular hypofunction, may also benefit from the compositions of the present invention. In the inner ear, the auditory organ houses the mechanically sensitive hair cells required to convert acoustic vibrations into electrical impulses. The vestibular organs, including semicircular canals (SSC), the ellipsoidal sac and the saccule, also contain sensory hair cells to detect head position and head motion. The compositions of the invention may be used, for example, in infusion; in a matrix or other system with long-lasting drug properties; or in other ear topical applications that enhance auditory reproduction.

anti-LRP 5/6 antibodies or antigen-binding fragments thereof (e.g., Wnt substitutes) may also be used for regeneration of retinal tissue. In adult mammalian retinas, Muller glia cells (Muller glia cells) are capable of regenerating retinal cells (including photoreceptors), e.g., following in vivo neurotoxic injury. Wnt signaling and enhancers of Wnt signaling can promote proliferation of mueller glial-derived retinal progenitor cells after damage or during degeneration. The compositions of the present invention may also be used to regenerate tissues and other cell types in the eye. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, keratopathy, fukes' dystrophy, vitreoretinopathy, genetic diseases, and the like may benefit from the compositions of the present invention. AMD is characterized by progressively reduced central vision and visual acuity. Fukes' dystrophy is characterized by a gradual loss of corneal endothelial cells. Wnt signaling and enhancement of Wnt signaling can promote regeneration of corneal endothelium, retinal epithelium, and the like in ocular tissues. In other embodiments, the compositions of the present invention may be used, for example, in infusion; in a matrix or other system with long-lasting drug properties; or in other ocular topical applications for the treatment of retinal regeneration and macular degeneration.

Specific proliferative cell populations for homeostatic renewal of hepatocytes, such as Axin2 positive cells in the pericentral region, have been identified by lineage tracing studies. Lineage tracing studies have also identified additional potential liver progenitor cells, including but not limited to Lgr positive cells. Self-renewing hepatocytes and other potential progenitor cell populations, including Lgr 5-positive cells and Axin 2-positive cells, were identified as capable of regenerating in response to Wnt signaling and/or R-spinal protein following injury. Many preclinical models of acute liver injury and failure, as well as chronic liver disease, show that recovery and regeneration of hepatocytes benefit from enhanced Wnt signaling. The compositions of the invention may be used for: treating acute liver failure and acute alcoholic liver injury; treating chronic liver disease, chronic alcoholic liver disease, alcoholic hepatitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) undergoing hepatitis C or hepatitis B virus infection or post-viral drug therapy; treatment of cirrhosis and severe chronic liver disease of all origins; and enhancing hepatocyte regeneration. Methods for regenerating liver tissue benefit from administration of a compound of the invention, which may be systemic or localized. These methods include, but are not limited to, systemic administration methods and localized administration methods, such as by injection into liver tissue, by injection into veins or blood vessels leading to the liver, by implantation of slow release formulations, and the like.

Wnt signaling plays an important role in the regeneration of various epithelial tissues. Various epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when rapidly dividing epithelial cells attached to the gastrointestinal tract rupture, rendering mucosal tissue susceptible to ulceration and infection. The part of the epithelial lining that covers the oral cavity, known as the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. Oral mucositis is probably the most common debilitating complication of cancer therapy, particularly chemotherapy and radiation. In addition, the compositions of the present invention may also be beneficial in the treatment of short bowel syndrome, Inflammatory Bowel Disease (IBD) or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving tooth, nail, or skin hypoplasia, and the like. The molecules of the invention may be used in all of these conditions in which regenerative cells are contacted with the compounds of the invention. Methods for regenerating epithelial tissue benefit from administration of a compound of the invention, which may be systemic or localized. The contacting may be, for example, topical, including intradermal, subcutaneous, in the form of a gel, lotion, cream, etc., for application at the targeted site, etc.

In addition to skin and gastrointestinal tract, Wnt signaling, as well as enhancement and promotion of Wnt signaling, also play important roles in the repair and regeneration of tissues, including pancreas, kidney, and lung, in preclinical models. anti-LRP 5/6 antibodies or antigen-binding fragments thereof (e.g., Wnt substitutes) may be beneficial for various diseases involving exocrine and endocrine pancreas, kidney, or lung. An anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., a Wnt substitute) can be used to treat metabolic syndrome; treating diabetes; treating acute or chronic pancreatitis, exocrine pancreatic insufficiency; treating acute kidney injury and chronic kidney disease; treating pulmonary diseases includes, but is not limited to, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, particularly Idiopathic Pulmonary Fibrosis (IPF), and other conditions that result in loss of lung epithelial tissue. Methods for regenerating these tissues benefit from administration of the compounds of the invention, which may be systemic or localized.

Epidermal Wnt signaling, coordinated with signaling via other developmental factors, is critical for adult hair follicle regeneration. Alopecia is a common problem, and androgenetic alopecia, commonly known as male pattern baldness, is the most common form of hair loss in men. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive population of cells with a molecule of the invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g., a topical lotion, gel, cream, etc.

Stroke, traumatic brain injury, alzheimer's disease, multiple sclerosis, and other conditions affecting the Blood Brain Barrier (BBB) may be treated with an anti-LRP 5/6 antibody or antigen binding fragment thereof (e.g., Wnt substitute). Angiogenesis is critical to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is especially important for the CNS, since nervous tissue is extremely sensitive to hypoxia and ischemia. CNS endothelial cells that form the BBB differ from endothelial cells in non-neural tissues in that CNS endothelial cells are highly polarized cells held together by tight junctions and express specific transporters. Wnt signaling regulates CNS angiogenesis and/or function. BBB impaired conditions may benefit from administration of the compounds of the invention, which may be systemic or localized, e.g., by direct injection, intrathecal administration, implantation of slow release formulations, and the like. In addition, Wnt signaling is actively involved in neurogenesis and plays a neuroprotective role after injury. The compositions of the present invention may also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and the like.

Wnt signaling also plays a role in angiogenesis. anti-LRP 5/6 antibodies or antigen binding fragments thereof (e.g., Wnt substitutions) may be beneficial in the treatment of conditions in which angiogenesis is beneficial, myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, and the like, as well as conditions from genetic diseases. Methods for regenerating these tissues benefit from administration of the compounds of the invention, which may be systemic or localized.

In certain embodiments, the methods of the invention promote tissue regeneration, for example in tissue that is subject to damage or tissue or cell reduction or loss. The loss or damage can be anything that results in a reduction in the number of cells, including disease or injury. For example, accidents, autoimmune disorders, treatment side effects or disease conditions may constitute trauma. Tissue regeneration increases the number of cells within the tissue and preferably enables the cells of the tissue to reestablish connections and more preferably enables the tissue to regain function.

Reduction of Wnt pathway signaling and related therapeutic methods

In certain embodiments, an anti-LRP 5/6 antibody or antigen binding fragment thereof can be used to reduce or inhibit Wnt signaling in a tissue or cell. Thus, in some aspects, the invention provides a method for reducing Wnt signaling or inhibiting Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof disclosed herein, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In particular embodiments, the cell is a cultured cell and the contacting occurs in vitro.

In a related aspect, the invention provides a method for reducing or inhibiting Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising an anti-LRP 5/6 antibody or antigen-binding fragment thereof of the invention, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a 5 'cap sequence and/or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to a coding sequence.

In a related aspect, the invention provides a method for reducing or inhibiting Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the vector is an expression vector and may include a promoter sequence operably linked to a nucleic acid sequence. In particular embodiments, the vector is a viral vector.

In a related aspect, the invention provides a method for reducing or inhibiting Wnt signaling in a tissue, the method comprising contacting the tissue with an effective amount of a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In particular embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In certain embodiments, the cell is transduced with a vector comprising an expression cassette encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In particular embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

anti-LRP 5/6 antibodies and antigen-binding fragments thereof, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is an antagonist or inhibitor of the Wnt signaling pathway, can be used to treat a disease, disorder, or condition, e.g., by reducing or inhibiting Wnt signaling in a cell, tissue, or organ. Thus, in some aspects, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with increased or deregulated Wnt signaling or a disease or disorder for which reducing Wnt signaling would provide a therapeutic benefit) in a subject in need thereof, comprising contacting the subject with an effective amount of a composition comprising an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the anti-LRP 5/6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In particular embodiments, the composition is a pharmaceutical composition comprising any one of the following: an anti-LRP 5/6 antibody or antigen-binding fragment thereof; a polynucleotide comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, e.g., DNA or mRNA, optionally modified mRNA; a vector, e.g., an expression vector or a viral vector, comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof; or a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, e.g., a cell transduced with an expression vector or viral vector encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof. In particular embodiments, the disease or condition is a pathological disease or disorder or injury. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to the subject.

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with increased Wnt signaling or a disease or disorder for which reducing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor disclosed herein. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a 5 'cap sequence and/or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to a coding sequence.

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with increased Wnt signaling or a disease or disorder for which reducing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the vector is an expression vector and may include a promoter sequence operably linked to a nucleic acid sequence. In particular embodiments, the vector is a viral vector.

In a related aspect, the invention provides a method for treating a disease or condition (e.g., a disease or disorder associated with increased Wnt signaling or a disease or disorder for which reducing Wnt signaling would provide a therapeutic benefit), comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a cell comprising a nucleic acid sequence encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In particular embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding an anti-LRP 5/6 antibody or antigen-binding fragment thereof. In particular embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

In certain embodiments, methods of treating or preventing a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-LRP 5/6 antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is an inhibitor of the Wnt signaling pathway, can be used to treat a cancer or tumor, e.g., a solid tumor or a liquid tumor. Examples of cancers and tumors that may be treated include, but are not limited to: colon tumors (e.g., colon cancer or adenoma), stomach tumors (e.g., gastric cancer), small intestine tumors (e.g., small intestine cancer), liver tumors (e.g., liver cancer), pancreas tumors (e.g., pancreatic cancer), lung tumors (e.g., lung cancer), ovary tumors (e.g., ovarian cancer), kidney tumors (e.g., kidney cancer), brain tumors (e.g., brain cancer), spinal cord tumors (e.g., spinal cord cancer), skin tumors (e.g., skin cancer or melanoma), head and neck tumors (e.g., head and neck cancer), gastrointestinal tumors (e.g., gastrointestinal tract cancer, esophageal cancer, oral mucosal cancer, tongue cancer, stomach cancer, intestinal cancer, colon cancer), breast tumors (e.g., breast cancer), prostate tumors (e.g., prostate cancer), bone tumors (e.g., bone cancer), vascular tumors, Wilms tumors, leukemia/lymphoma, soft tissue tumors (e.g., soft tissue sarcoma or synovial sarcoma), metastatic cancer, and the like.

In certain embodiments, methods of treating or preventing a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is an inhibitor of the Wnt signaling pathway, may be used to treat a degenerative disease. Examples of degenerative diseases that may be treated include, but are not limited to, osteoarthritis, cartilage degeneration, sports injuries (e.g., cartilage damage), retinopathy, atherosclerosis, neurodegenerative disorders, and vascular disorders, e.g., vasculitis, conditions of abnormal angiogenesis.

In certain embodiments, methods of treating or preventing a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-LRP 5/6 antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is an inhibitor of the Wnt signaling pathway, may be used to treat fibrosis. Examples of fibrosis that can be treated include, but are not limited to, pulmonary fibrosis (including, but not limited to, COPD, idiopathic pulmonary fibrosis), renal fibrosis (e.g., end stage renal failure), liver fibrosis, congenital liver storage disease, and cardiac fibrosis.

In certain embodiments, the methods of treating or preventing a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is an inhibitor of the Wnt signaling pathway, can be used to treat heart failure (e.g., congestive heart failure, systolic heart failure, preserved ejection fraction heart failure), or coronary artery disease.

In certain embodiments, methods of treating or preventing a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-LRP 5/6 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds an inhibitor of the Wnt signaling pathway, may be used to treat ectopic ossification, osteopetrosis, or a congenital high bone mass disorder.

As used herein, the term "administering" or "introducing" or "providing" refers to the delivery of a composition to a cell, tissue and/or organ of a subject, or to a subject. Such administration or introduction may occur in vivo, in vitro, or ex vivo.

In particular embodiments, the pharmaceutical composition is administered parenterally, e.g., intravenously, orally, rectally, or by injection. In some embodiments, the pharmaceutical composition is administered topically (locally), e.g., topically (topically) or intramuscularly. In some embodiments, the composition is administered to a target tissue, such as a bone, a joint, an ear tissue, an eye tissue, a gastrointestinal tract, skin, a wound site, or a spinal cord. The methods of the invention may be practiced in vivo or ex vivo. In some embodiments, contacting the target cell or tissue with the tissue-specific Wnt signaling enhancing molecule is performed ex vivo, followed by implantation of the cell or tissue, e.g., an activated stem cell or progenitor cell, into the subject. The skilled artisan can determine the appropriate site and route of administration based on the disease or condition being treated.

The dosage and dosage regimen may depend on a variety of factors that are readily determinable by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's medical history. In particular embodiments, the amount of anti-LRP 5/6 antibody or antigen-binding fragment thereof (e.g., Wnt substitute) administered or provided to the subject ranges from about 0.01mg/kg to about 50mg/kg, 0.1mg/kg to about 500mg/kg, or about 0.1mg/kg to about 50mg/kg of the subject's body weight.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, e.g., reducing the likelihood of developing the disease or symptoms thereof in a subject, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects caused by the disease. As used herein, "treatment" covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., causing regression of the disease. A therapeutic agent (e.g., an anti-LRP 5/6 antibody or antigen-binding fragment thereof) can be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of developing diseases, wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms. It is desirable to perform such treatment before the affected tissue is completely functionally lost. Ideally, the subject treatment will be administered during and in some cases after the symptomatic phase of the disease. In some embodiments, the subject methods result in therapeutic benefits, such as preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, and the like. In some embodiments, the subject methods include the step of detecting that a therapeutic benefit has been achieved. One of ordinary skill in the art will appreciate that such a measure of treatment efficacy will be appropriate for the particular disease being modified, and will recognize appropriate detection methods for measuring treatment efficacy.

Promoting growth of cells, tissues and organoids and related methods

Other embodiments relate, in part, to the use of the Wnt-substitute molecules disclosed herein to promote or enhance the growth or proliferation of cells, tissues and organoids, e.g., by contacting the cells or tissues with one or more Wnt-substitutes, optionally in combination with a Norrin or rsspondin polypeptide. In certain embodiments, the cell or tissue is contacted ex vivo, in vitro, or in vivo. Such methods may be used to produce cells, tissues or organoids for therapeutic use, e.g., implantation or transplantation thereof into a subject. They may also be used to produce cells, tissues or organoids for research use. Wnt-replacement molecules have wide application in non-therapeutic approaches, such as in vitro research methods.

The invention provides a method for tissue regeneration of damaged tissue, such as the tissue described above, comprising administering to a cell a Wnt-substituting molecule. The Wnt-substitute molecule can be administered directly to the cell in vivo, orally, intravenously or by other methods known in the art to the subject or to cells ex vivo. In some embodiments where the Wnt-substitute molecule is administered to cells ex vivo, the cells may be transplanted into the subject before, after, or during administration of the Wnt-substitute molecule.

Wnt signaling is a key component of stem cell culture. For example, stem cell culture media described in WO2010/090513, WO2012/014076, Sato et al, 2011 (GASTROENTEROLOGY) 2011; 141: 1762-. The Wnt-replacement molecules disclosed herein are suitable alternatives to Rspondin for use in these stem cell media, or may be combined with rsponin.

Thus, in one embodiment, the present disclosure provides a method for enhancing the proliferation of a stem cell, the method comprising contacting a stem cell with one or more Wnt-surrogate molecules disclosed herein. In one embodiment, the present disclosure provides a cell culture medium comprising one or more Wnt-substituted molecules disclosed herein. In some embodiments, the cell culture medium can be any cell culture medium known in the art that typically includes Wnt or Rspondin, but in which Wnt or Rspondin is replaced (in whole or in part) or supplemented with one or more Wnt-replacement molecules disclosed herein. For example, the culture medium may be one as described in WO2010/090513, WO2012/014076, Sato et al, 2011 (gastroenterology 2011; 141: 1762-.

The stem cell culture medium typically includes additional growth factors. Thus, the method may further comprise supplying a growth factor to the stem cells. Growth factors commonly used in cell culture media include epidermal growth factor (EGF, (Peprotech)), transforming growth factor-alpha (TGF-alpha, pepertak), basic fibroblast growth factor (bFGF, pepertak), brain-derived neurotrophic factor (BDNF, R & D system), Hepatocyte Growth Factor (HGF), and keratinocyte growth factor (KGF, pepertak, also known as FGF 7). EGF is a potent mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has profound effects on the differentiation of specific cells in vivo and in vitro, as well as certain fibroblasts in cell culture. Mitogenic growth factors may be added to the medium every two days, with the medium preferably being refreshed every four days. Typically, the mitogenic factor is selected from: i) EGF, TGF-alpha and KGF, ii) EGF, TGF-alpha and FGF 7; iii) EGF, TGF-alpha and FGF; iv) EGF and KGF; v) EGF and FGF 7; vi) EGF and FGF; vii) TGF-alpha and KGF; viii) TGF-alpha and FGF 7; or ix) TGF-alpha and FGF. In certain embodiments, the disclosure encompasses a stem cell culture medium comprising, for example, the Wnt-substitute molecules disclosed herein, optionally in combination with one or more of the growth factors described herein, or a combination thereof.

These methods of enhancing stem cell proliferation can be used to grow new organoids and tissues from stem cells, as described, for example, in WO2010/090513, WO2012/014076, Sato et al, 2011 (gastroenterology 2011; 141: 1762-.

In some embodiments, the Wnt-substituted molecule is used to enhance stem cell regeneration. Illustrative stem cells of interest include, but are not limited to: a muscle satellite cell; hematopoietic stem cells and progenitor cells derived therefrom (U.S. patent No. 5,061,620); neural stem cells (see Morrison et al (1999) Cell (Cell) 96: 737-749); embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; hepatic stem cells; adipose tissue-derived stem cells, and the like.

Diagnostics and related methods

Other embodiments of the invention relate, in part, to diagnostic applications for detecting the presence of cells or tissues expressing LRP5 and/or LRP 6. Accordingly, the present disclosure provides methods of detecting LRP5 and/or LRP6 (e.g., detecting cells or tissues expressing LRP5 or LRP 6) in a sample. Such methods may be applied in a variety of known detection formats, including but not limited to Immunohistochemistry (IHC), Immunocytochemistry (ICC), In Situ Hybridization (ISH), whole-set in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, Enzyme Immunoassay (EIA), and enzyme-linked immunoassay (ELISA). In particular embodiments, the methods comprise contacting a tissue or cell, e.g., obtained from a subject, with an antibody or antigen-binding fragment thereof disclosed herein, and then determining the amount of binding of the antibody or antigen-binding fragment thereof to the tissue or cell, thereby determining the presence or amount of one or more LRP5 and/or LRP6 receptors in the tissue or cell.

ISH is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., primary binding agents) to localize specific DNA or RNA sequences in a portion or segment of a cell or tissue (in situ), or if the tissue is small enough, in the entire tissue (self-contained ISH). One of ordinary skill in the art will recognize that this is in contrast to immunohistochemistry, which uses antibodies as the primary binding agent to localize proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes against genomic DNA. Fluorescent dnaish (fish) may be used, for example, in medical diagnostics to assess chromosomal integrity. RNA ISH (hybrid histochemistry) is used to measure and locate mRNA and other transcripts in tissue sections or packages.

In various embodiments, the antibodies and antigen-binding fragments thereof described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, an antibody "conjugate" refers to an anti-LRP 5/6 antibody or antigen-binding fragment thereof covalently linked to a detectable label. In the present invention, the DNA probe, RNA probe, monoclonal antibody, antigen-binding fragment thereof and antibody derivatives thereof, such as single-chain variable fragment antibody or epitope-labeled antibody, may all be covalently linked to a detectable label. In "direct detection", only one detectable antibody, i.e., the first detectable antibody, is used. Thus, direct detection means that an antibody itself conjugated to a detectable label can be detected without the addition of a second antibody (secondary antibody).

A "detectable label" is a molecule or material that can produce a detectable (e.g., visual, electronic, or otherwise) signal indicative of the presence and/or concentration of the label in a sample. When conjugated to an antibody, a detectable label can be used to locate and/or quantify the target to which the specific antibody is directed. Thus, the presence and/or concentration of the target in the sample can be detected by detecting the signal generated by the detectable label. The detectable label may be detected directly or indirectly, and several different detectable labels conjugated to different specific antibodies may be used in combination to detect one or more targets.

Examples of detectable labels that can be directly detected include fluorescent dyes and radioactive substances as well as metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., a second antibody, after the application of the first antibody. Thus, detection is performed by detecting the binding of the second antibody or binding agent to the first detectable antibody. Examples of first detectable binding agents or antibodies that require the addition of a second binding agent or antibody include enzyme detectable binding agents and hapten detectable binding agents or antibodies.

In some embodiments, the detectable label is conjugated to a nucleic acid polymer comprising a first binding agent (e.g., in an ISH, WISH, or FISH process). In other embodiments, the detectable label is conjugated to an antibody comprising a first binding agent (e.g., during IHC).

Examples of detectable labels that may be conjugated to the antibodies used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5- (and 6) -carboxyfluorescein, 5-or 6-carboxyfluorescein, 6- (fluorescein) -5- (and 6) -carboxamidohexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine and dyes (such as Cy2, Cy3 and Cy5), optionally substituted coumarins (including AMCA, PerCP), phycobiliproteins (including R-phycoerythrin (RPE) and Allophycocyanin (APC)), texas Red, prinston Red (Princeton Red), Green Fluorescent Protein (GFP) and analogues thereof, as well as conjugates of R-phycoerythrin or allophycocyanin, inorganic fluorescent labels, such as particles based on semiconductor materials, such as coated CdSe nanocrystals.

Examples of polymer particle labels include microparticles or latex particles of polystyrene, PMMA or silica that can be embedded with a fluorescent dye or polymer micelles or capsules containing a dye, enzyme or substrate.

Examples of metal particle labels include gold particles that can be converted by silver staining and coated gold particles. Examples of haptens include DNP, Fluorescein Isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), beta-Galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, beta-glucuronidase, invertase, xanthine oxidase, firefly luciferase and Glucose Oxidase (GO). Examples of commonly used substrates for horseradish peroxidase include 3,3' -Diaminobenzidine (DAB), diaminobenzidine with nickel-enhancing action, 3-amino-9-ethylcarbazole (AEC), Benzidine Dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indigo (IB), Tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphthol pyronine (alpha-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2- (p-iodophenyl) -3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), 5-bromo-4-chloro-3-indolyl-beta-D-galactoside/iron galactoside Ferricyanide (BCIG/FF).

Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B1-phosphate/fast red TR (NABP/FR), naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), naphthol-AS-B1-phosphate/-fast red TR (NABP/FR), naphthol-AS-MX-phosphate/fast TR (NAMP/FR), naphthol-AS-B1-phosphate/neofuchsin (NABP/NF), bromochloroindole phosphate/nitro blue tetrazolium (BCIP/NBT), 5-bromo-4-chloro-3-indolyl-B-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridinium ester, 1, 2-dioxetane, and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioisotopes of iodine, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

The detectable label may be linked to an antibody described herein or any other molecule that specifically binds to a biomarker of interest, such as an antibody, nucleic acid probe, or polymer. Furthermore, one of ordinary skill in the art will appreciate that the detectable label may also be conjugated to a second and/or third and/or fourth and/or fifth binding agent or antibody or the like. Furthermore, the skilled person will understand that each additional binding agent or antibody used to characterize the biological marker of interest may be used as a signal amplification step. The biomarkers can be detected visually using, for example, light microscopy, fluorescence microscopy, electron microscopy, wherein the detectable substance is, for example, a dye, a colloidal gold particle, a luminescent reagent. The visually detectable substance bound to the biomarker can also be detected using a spectrophotometer. When the detectable substance is a radioisotope, it may be detected visually by autoradiography, or non-visually using a scintillation counter. See, e.g., Larsson,1988, "immunocytochemistry: theory and Practice (Immunocytochemistry: the institution and Practice) (CRC Press, Boca Raton, Fla., Po.) of Poka Lalton, Florida); methods in Molecular Biology (Methods in Molecular Biology), volume 80, 1998, John d.pound (eds.) (Humana Press, Totowa, n.j., tokawa, nj.).

The invention further provides a kit for detecting one or more LRP receptors or cells or tissues expressing one or more LRP receptors in a sample, wherein the kit contains at least one antibody, polypeptide, polynucleotide, vector or host cell described herein. In certain embodiments, the kit can include buffers, enzymes, labels, substrates, beads or other surfaces, etc. associated with the antibodies of the invention, as well as instructions for use.

All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be restricted except in light of the attached claims.

Examples of the invention

Example 1

Characterization of anti-LRP 5/6 antibodies

The antibodies Fab, scFv and VHH or sdAb fragments disclosed herein were sequenced and subcloned into mammalian expression vectors for expression, purification, and characterization of binding affinities relative to various LRP receptors.

Soluble recombinant proteins were prepared according to the manufacturer's instructions by transfecting the corresponding expression vectors into Expi293F cells (Thermo Fisher Scientific, Waltham, MA). Briefly, four days after transfection, cell culture medium was collected after slowing down the cell pellet rotation. The medium was incubated with protein a resin (REPLIGEN, Waltham, MA) to collect proteins containing human IgG-Fc moieties, or with nickel affinity resin (Roche, Basel, Switzerland) to collect proteins conjugated to His-tag. The protein was eluted from the protein A resin with 10mM glycine, pH 3.5 or from the nickel affinity resin with 150mM imidazole, pH7.4, respectively.

Subsequently, the protein eluate is fractionated and further purified by Size Exclusion Chromatography (SEC). SEC was performed by flash protein liquid chromatography using Superdex200 Increate 10/300GL (GE Healthcare, Pittsburgh, Pa.) in HBS buffer (10mM HEPES, 150mM NaCl, pH 7.4). Each protein was injected onto the column in a volume of 475. mu.l or 500. mu.l. The absorbance at 280nm was monitored and 500 μ l fractions of all eluates were collected. Each collected fraction near the main peak was further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed using Tris-HCl 4-15% gels (Bio-Rad, Hercules, Calif.) under non-reducing and reducing conditions. Samples were prepared in Laemmli sample buffer and heated at 100 ℃ for 5 min.

Protein concentration was determined by direct UV a280 method using a NanoDrop spectrophotometer (seemer science and technology). The absorbance versus protein concentration is linear based on Beer-Lamber equation a-l c; a is the absorbance value, is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration. The experimental extinction coefficients of all the proteins produced were estimated by their amino acid sequences.

The binding kinetics of antibody fragments to LRP5 extracellular domain (LRP5 ECD) and/or LRP6 extracellular domain (LRP6ECD) protein targets was determined by biolayer interferometry (BLI), using an Octet Red 96 (PALLForteBio, Fremont, CA) instrument with a Streptavidin (SA) biosensor at 30 ℃, 1000 rpm. The C-terminal biotinylated LRP ECD recombinant protein was diluted to 20nM in running buffer (PBS, 0.05% tween-20, 0.5% BSA, pH 7.2) and the SA biosensor captured until the coupling length reached 0.2 nM. After capturing LRP5 or LRP6, SA biosensors with captured biotinylated LRP5 or biotinylated LRP6 were immersed in wells containing the relevant antibody fragments at 7 different concentrations (0nM, 1.37nM, 4.12nM, 12.4nM, 37nM, 111.1nM, 333.3nM, 1000nM) in running buffer and wells with running buffer as reference channel only. K _DDetermined by global fitting, 1:1 binding model, according to the manufacturer's suggested settings.

Binding measurements were also performed by performing surface plasmon resonance on BIAcore T100 (general electric medical group, pittsburgh, pennsylvania) and purifying all proteins by SEC prior to the experiment. Biotinylated Lrp6E3E4 was coupled at low density to streptavidin on an SA sensor chip (general electric medical group, pittsburgh, pa). Irrelevant biotinylated proteins were captured to the control flow cell at equivalent coupling densities. Increasing concentrations of scFv-Nab fusion molecules (e.g., 18R5 scFv-LRP6 bound Nab) 40. mu.l/ml 1xHBS-P (general electro-medical group, Pittsburgh, Pa.) containing 0.5% BSA were flowed through the chip. The chip surface was regenerated after each injection of 2M MgCl2 in HBS-P for 60 seconds. The reference values were subtracted from the curves and all data were analyzed using Biacore T100 evaluation software version 2.0 and 1:1Langmuir binding model to determine KD values.

Table 1A provides the heavy chain CDRs (CDRH1, CDRH2, and CDRH3) and light chain CDRs (CDRL1, CDRL2, and CDRL3) of the indicated antibody clones, and indicates that the antibody fragments are shown as the original LRP5 or LRP6 to which they bind. The Specificity Determining Regions (SDRs) shown below were mapped using the Abgenetics software from Distributed Bio, which contained the Kabat definition of the CDRs (Padlan et al, J. Am. Experimental biol., USA 9,133-139 (1995)).

In the case where no light chain CDRs are provided, the antibody fragment does not include a light chain.

Table 1A: cloning of ID and CDR sequences

Table 1B provides the sequence identifier numbers of antibody heavy chain fragments (HC) for an illustrative clone having only heavy chain regions, as well as the binding properties of the illustrative clone. In certain embodiments, the LRP5/6 binding domain is a Fab or derived from a Fab, thus table 1B comprises VH and CH1 sequences, but does not comprise CH2 or CH3 sequences. In certain embodiments, the LRP5/6 binding domain is or is derived from a VHH or sdAb, thus table 2B comprises VHH domains. K determined by Octet BLI is shown for various Fzd receptors_DAnd (4) data. The column "confirmed binding" indicates the binding result from Octet BLI binding or BIAcore SPR. Blank entries indicate that binding to a particular Fzd receptor has not been determined. As shown in table 1B, the Octet BLI or BIAcore SPR sensorgrams of anti-LRP 5/6 antibody fragments showed affinity ranges for LRP5 and/or LRP 6.

TABLE 1B clone ID, Heavy Chain (HC) Seq ID No and binding characteristics

Clone ID	HC Seq ID NO	BLI or SPR confirmed binding	Kd(nM)
				001S-F11	1	LRP6e1e2	*
009S-G02	2	LRP6e1e2	*
				009S-A03	3	LRP6e1e2	*
009S-D03	4	LRP6e1e2	*
				009S-F03	5	LRP6e1e2	*
009S-H03	6	LRP6e1e2	*
				009S-A04	7	LRP6e1e2	*
009S-B04	8	LRP6e3e4	*
				009S-D04	9	LRP6e3e4	**
009S-E04	10	LRP6e3e4	*
				009S-F04	11	LRP6e3e4	**
009S-G04	12	LRP6e3e4	*
				009S-H04	13	LRP6e3e4	*
009S-A05	14	LRP6e3e4	**
				013S-G04	15	LRP6e3e4	*
013S-H04	16	LRP6e3e4	*
				013S-C05	17	LRP6e3e4	*
013S-D05	18	LRP6e3e4	*
				013S-G04	19	LRP6e3e4	*
013S-H04	20	LRP6e3e4	*
				013S-A05	21	LRP6e3e4	**
013S-C05	22	LRP6e3e4	*
				013S-D05	23	LRP6e3e4	*
008S-D01	24	LRP5

Indicates <500 nM; indicate >500nM

Table 1C provides additional binding properties of certain clones, including relative binding affinities for LRP6E1E2 and LRP6E3E4 domains. The entry "n.b." indicates no binding.

TABLE 1C LRP6 binding characteristics of a subset of clones

Indicates <500 nM; indicate >500nM

Example 2

Alanine scanning mutation of anti-LRP 6 antibody fragment

One antibody fragment (009S-E04) was selected for alanine-scanning mutagenesis of the CDRs and the corresponding LRP6 binding affinity of the various mutants was determined by Octet BLI as described in example 1. As shown in table 2, the large number of mutants bound LRP6 with similar affinity as the wild-type antibody fragment indicates that the LRP6 antibody and antigen binding fragments thereof can tolerate amino acid modifications within the CDRs.

TABLE 2.009S-E04 alanine scanning mutant CDR sequences and K with biotinylated LRP6CRD as determined by Octet BLI_D(nM)。

Indicates <100nM

Example 3

293 and A375W_NT18R5 in a dependent reporter assay_SCFV-L_RPVHH W_NTCharacterization of surrogate fusion proteins

The ability of Wnt-substituted fusion proteins comprising a scFv region that binds to one or more Fzd and a VHH or sdAb (or single domain antibody, Nab) region that binds to LRP5 and/or LRP6 was demonstrated. Fzd conjugate 18R5 in scFv form was fused to various Lrp5 and Lrp6 conjugates disclosed herein. The LRP binder was Nab and was fused to the C-terminus of the 18R5 scFv via a 6-amino acid linker. The fusion protein containing the C-terminal multi-His-tag was expressed in expi293 cells according to the manufacturer's protocol and purified from conditioned medium using the intact His-tag purification resin and Superdex 20010/300 GL equilibrated in HBS (10mM HEPES, pH 7.3, 150mM NaCl). Fractions containing the monomer fusions were combined and concentrated.

The ability of the fusion proteins to induce Wnt pathway signaling was tested using the Wnt dependent reporter assay in 293 and a375 cell lines under the following conditions. 10,000A 375 and HEK293 Wnt reporter cells stably transfected with STF Wnt reporter plasmid or variant thereof were seeded in 96-well plates in triplicate in each case and stimulated with the fusion protein in the presence or absence of 25 nFc-Rspo 2 for 16-20 hours. The conditions tested included various concentrations of each fusion protein at 10^-2nM to 10³In nM. After washing the cells with PBS, the cells in each well were lysed in 30 μ l of passive lysis buffer (Promega). 10 μ l of lysate per well was measured using the Firefly Luciferase Assay (Firefo Luciferase Assay) kit (Promega).

The results indicate that many Wnt substitutes tested activated the Wnt signaling pathway in a concentration-dependent manner, and that it was further enhanced by treatment with Fc-Rspo2 (data not shown).

Example 4

L_RP6E3E4 Crystal Structure of conjugate complexes

Lrp6 (low density lipoprotein receptor-related protein 6) is a single-pass membrane protein containing 1613 amino acids that plays a key role in Wnt-mediated activation of β -catenin signaling. Lrp6 and related Lrp5 share 70% sequence identity between them. The extracellular regions of Lrp6 and Lrp5 contain four beta propeller domains referred to herein as E1, E2, E3, and E4 domains. In this section, the structure of the Lrp6E3E4 domain is depicted (residues 631 to 1246 of uniprot entries O75581 (https:// www.uniprot.org/uniprot/O75581)). The sequence Lrp6E3E4 construct containing the biotin receptor peptide (BAP) and the octa-histidine motif at its C-terminus for structural studies is shown below:

LRP6E3E4_O75581_631-1246：

EAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDFDVTDNRIYWTDISLKTISRAFMNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNRIEVSKLDGQHRQVLVWKDLDSPRALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTLVPNVGRANGLTIDYAKRRLYWTDLDTNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIYWTDWSRRSIERANKTSGQNRTIIQGHLDYVMDILVFHSSRQSGWNECASSNGHCSHLCLAVPVGGFVCGCPAHYSLNADNRTCSAPTTFLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVRAIDYDPLDKQLYWIDSRQNMIRKAQEDGSQGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYWTCEATNVINVTRLDGRSVGVVLKGEQDRPRAVVVNPEKGYMYFTNLQERSPKIERAALDGTEREVLFFSGLSKPIALALDSRLGKLFWADSDLRRIESSDLSGANRIVLEDSNILQPVGLTVFENWLYWIDKQQQMIEKIDMTGREGRTKVQARIAQLSDIHAVKELNLQEYRQHPCAQDNGGCSHICLVKGDGTTRCSCPMHLVLLQDELSCGEPPSGSGGLNDIFEAQKIEWHEGSGSHHHHHHHH(SEQ ID NO:619)

Example 5

L for structural studies_RPExpression and purification of 6E3E4

A stable cell line expressing Lrp6E3E4 domain was generated in Expi293 cells with G418 selection. For large scale expression, frozen vials of Expi293 cells expressing Lrp6E3E4 were thawed to 20mL Expi293 medium (Thermofeisher)In (1). The viability of the cells is monitored and the cells are consumed every other day until a desired volume (typically 6L to 10L) is reached

To 4.0 × 10⁶Density of individual cells/mL. At this stage, cells treated with 2mM valproic acid were grown continuously to higher densities and at

The medium was harvested by centrifugation after hours. Fzd CRD _ Xtal protein was purified from the culture medium by incubation with HisComplete resin (1 mL per L culture; Roche) pre-equilibrated in PBS (50mM sodium phosphate monobasic pH 8.0, 300mM NaCl) and eluted with 250mM imidazole. The eluate was concentrated to 5mL and further refined on a HiLoad 16/600Superdex 200pg column (GE Life Sciences) pre-equilibrated with HBS (20mM HEPES pH 7.4 and 150mM sodium chloride). Fractions near the main peak were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the content. SDS-PAGE was performed under non-reducing conditions using Tris-HCl 4-15% gel (Burley, Heracles, Calif.). Samples were prepared in Laemmli sample buffer and heated at 100 ℃ for 5 minutes. The fraction containing LRP6E3E4 was concentrated to

And frozen in the presence of 10% glycerol for storage at-80 ℃ until further use. Protein concentration was determined by direct UV a280 method using a NanoDrop spectrophotometer (seemer science and technology). The absorbance versus protein concentration is linear based on Beer-Lamber equation a-l c; a is the absorbance value, is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration. The extinction coefficients of all the produced proteins were estimated by their amino acid sequences.

Example 6

Expression and purification of VHH or sdAb or Fab conjugates

Following from the manufacturer (Saimer fly)El), plasmids expressing light and heavy chains (with a hexa-histidine tag at their C-terminus) in the case of Fab or VHH conjugates were transfected for expression in Expi293 cells, typically on a 1000mL scale. After 4 days of continuous cell growth, the medium was harvested by centrifugation and combined with Complete-His resin pre-equilibrated in PBS (2.5 mL per 1L of culture; Roche) and eluted under gravity flow using 250mM imidazole in PBS. Concentrating the eluate containing Fab-conjugates to

And further refined on a HiLoad 16/600Superdex 200pg column (universal electrical life sciences) pre-equilibrated with HBS. Fractions near the main peak were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the content. SDS-PAGE was performed under non-reducing conditions using Tris-HCl 4-15% gels (Burley, Heracles, Calif.). Samples were prepared in Laemmli sample buffer and heated at 100 ℃ for 5 minutes. Concentrating the fraction containing Fab or VHH conjugates to

And frozen in the presence of 10% glycerol for storage at-80 ℃ until further use. Protein concentration was determined by direct UV a280 method using a NanoDrop spectrophotometer (seemer science and technology). The absorbance versus protein concentration is linear based on Beer-Lamber equation a-l c; a is the absorbance value, is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration. The extinction coefficients of all the produced proteins were estimated by their amino acid sequences.

Example 7

L_RP6E3E4:VHH/F_ABComplex formation, crystallization and structure determination

The purified Lrp6E3E4 and VHH/Fab conjugate were mixed at a molar ratio of 1.1:1 (slightly in excess relative to the smaller molecular weight protein) and incubated with carboxypeptidases a and B at a w/w ratio of 100:1 at 4 ℃ overnight. Complex formation was confirmed by the observation of a single major peak on a superdex s200 Increase (10/300GL) column pre-equilibrated in HBS. The fractions containing the complexes were further examined by SDS-PAGE and concentrated to 10mg/mL to 25mg/mL for crystallization screening. Initial crystallization screens (using commercially available MCSG1, MCSG2, MCSG3, MCSG4, PACT (molecular size)), PEGI and PEG II (qiagen) screens and optimization by grid screening or microspecies matrix screening [ MMS; optimized microcrystalline seed matrix screening for protein crystallization: what did we learn? (microscopic matrix screening for optimization in protein crystallization. The 96-well plate crystal screening experiments were monitored manually at regular intervals by a discover v20 stereomicroscope (Zeiss) and data were collected by freezing the crystals by plunging them into liquid nitrogen in the presence of various cryoprotectants, typically 15% to 30% v/v glycerol or ethylene glycol or 1.1M to 2.5M sodium malonate (sodium malanate) pH 7.0. The X-ray diffraction data set was collected at the Berkeley Structural Biology Center for Advanced Light Sources (ALS) of Berkeley, Calif. and processed using the programs XDS [ Kabsch, W.XDS. [ Crystal science, D66,125, 132(2010) ] and xdsme [ Legend, P.XDSME: XDS Made easer (2017) GitHub library, https:// github.com/legrandp/xdsme DOI 10.5281/zenodo.837885 ]. The structure of the Lrp6E3E4 VHH/Fa complex was determined by: the molecular replacement method was performed using a pharmaci [ pharmaci crystallography software.a.j.mccoy, r.w.grosse-Kunstleve, p.d.adams, m.d.win, l.c.storoni, and r.j.read, journal of applied crystallography (J Appl crystallography) 40, 658-: 4A 0P; chen S, Bubeck D, MacDonald BT, Liang WX, Mao JH, malinauskasas T, LlorcaO, Aricescu AR, Siebold C, He X, Jones EY. developmental cells (dev. cell) 21848-61 (2011), and VHH or sdAb [ PDB code: 6B20, chain E; the published structures of natural communication (Nat Commun) 91996 (2018), Gulati S, Jin H, Masuho I, Orban T, Cai Y, Pardon E, Martemonanov KA, Kiser PD, Stewart PL, Ford CP, Steyaert J, Palczewski K, then refined and validated by MolProbity, as implemented in Phenix [ PHENIX: python-based integrated system for macromolecular structure solutions p.d.adams, p.v.afonine, g.bunkoczi, v.b.chen, i.w.davis, n.echos, j.j.head, l.w.hung, g.j.kapral, r.w.grosse-Kunstleve, a.j.mccoy, n.w.morarty, r.oeffner, r.j.2010, d.richardson, j.s.richardson, t.c.terwitger and p.h.zwart. crystal science D66, 213; MolProbity: all-atomic structure verification for macrocrystallography (MolProbity: all-atom structural validation for macrogolula), v.b.chen, w.b.arendall, j.j.headd, d.a.keedy, r.m.immrmino, g.j.kapral, l.w.murray, j.s.richardson, and d.c.richardson, crystal proceedings D66,12-21 (2010). The crystallographic model was examined manually and was built using COOT [ Features and development of COOT (Features and depth of COOT), p.emsley, b.lohkamp, w.g.scott and k.cowtan. Fine crystal structure analysis and image creation were performed using moe (ccg) and pymol (schrodinger).

Example 8

L_RPStructure of 6E3E4 VHH26 Complex

VHH26(009S-E04) sequence:

DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYADSVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSSGSGSGHHHHHH(SEQ ID NO:620)

diffraction-quality crystals of Lrp6E3E4: VHH26 (concentration 12.4mg/mL) were obtained by MMS in a PACT sieve under G8 conditions containing 0.2M sodium sulfate, 0.1M bis-Tris-propane pH 7.5 and 20% (v/v) of PEG 3350. The crystals were cryoprotected using a 27% glycerol pore solution. Lrp6E3E4 VHH26 complex in P3121 space group (

And is

) Wherein each asymmetric unit has one complex molecule. The Lrp6E3E4 VHH26 complex has the structure

Is determined at resolution and perfected to R_crystFactor and R_freeThe factors were 18.5% and 23.0%, respectively.

The overall structure of the Lrp6E3E4 VHH26 complex is shown in FIG. 3.1(A), which reveals that the epitope on Lrp6E3E4 directed against VHH26 (FIG. 4.1(B)) is located within the E3 β propeller of Lrp6 the structure of the complex allows the identification of an epitope on Lrp6E3E4 directed against VHH26 with the following residues of Lrp6E3E4 defining the core interaction site (the interatomic distance between Lrp6E3E4 and VHH26 is less than or equal to

)：

Arg639, Ala640, Lys622, Glu663, Ile681, Ser682, Lys684, Asp705, Tyr706, Glu708, Thr724, Gly725, Arg751, Trp767, Gly768, Gly769, Arg792, Leu810, Asp811, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875 and Met 877.

In addition, the following residues on Lrp6E3E4 can be identified as immediate interaction sites (the interatomic distance between Lrp6E3E4 and VHH26 is greater than

And is less than or equal to

)：

Arg638, Asp641, Val661, Ala664, Ser665, Asp680, Leu683, Thr685, Leu704, Pro707, Asp723, Thr726, Asn727, Asp748, Ser749, Pro750, Lys770, Pro771, Gly791, Asn794, Asp809, Thr812, Asn813, Pro833, Pro835, Asp849 and Arg 852.

The structure of the Lrp6E3E4 VHH26 complex allows the identification of the following residues on VHH26 as core interaction sites (the interatomic distance between Lrp6E3E4 and VHH26 is less than or equal to

)：

Gly26, Arg27, Phe29, Ala30, Ile31, Tyr32, Arg52, Pro53, Val54, Val55, Glu57, Asn74, Ala75, Lys77, Arg100, Pro101, Trp102, Gly103, Ser104, Arg105 Asp106 and Tyr 108.

The structure of the Lrp6E3E4 VHH26 complex also allows identification of the following residues on VHH26 as core interaction sites (the interatomic distance between Lrp6E3E4 and VHH26 is greater than that of VHH26

And is less than or equal to

)：

Gly24, Ser25, Ile28, Asp33, Met50, Ile51, Thr56, Arg72, Asn73, Met76, Lys99 and Glu 107.

Example 9

L_RPStructure of 6E3E4 VHH36 Complex

VHH36(013S-D05) sequence:

QVKLEESGGGLVQAGGSLRLSCAASGRIFSIYDMGWFRQAPGKEREFVSGIRWSGGTSYADSVKGRFTISKDNAKNTIYLQMNNLKAEDTAVYYCGSRGYWGQGTLVTVSSGSGSGHHHHHH(SEQ ID NO:621)

Diffraction-quality crystals of Lrp6E3E4: VHH36 (concentration 14.0mg/mL) were obtained by grid-screening optimization in the presence of 1.6M ammonium sulfate and 0.1M Tris pH 8.2. The crystals were cryoprotected using 1.1M sodium malonate pH 7.0 and Tris pH 9.0 in 1.6M ammonium sulfate. Lrp6E3E4 VHH36 Complex at P6₅Space group (

And is

) Wherein each asymmetric unit has one complex molecule. The Lrp6E3E4 VHH36 complex has the structure

Is determined at resolution and perfected to R_crystFactor and R_freeThe factors were 18.6% and 22.8%, respectively.

The overall structure of the Lrp6E3E4 VHH36 complex is shown in FIG. 3.1(A), which reveals that the epitope on Lrp6E3E4 directed against VHH36 (FIG. 4.1(B)) is located within the E3 β propeller of Lrp6 the structure of the complex allows the identification of an epitope on Lrp6E3E4 directed against VHH36 with the following residues of Lrp6E3E4 defining the core interaction site (the interatomic distance between Lrp6E3E4 and VHH36 is less than or equal to

)：

Glu663, Ser665, Ile681, Tyr706, Glu708, Thr724, Ser749, Arg751, Trp767, Gly768, Arg792, Leu810, Asn813, Pro833, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875, and Met 877.

In addition, the following residues on Lrp6E3E4 can be identified as immediate interaction sites (the interatomic distance between Lrp6E3E4 and VHH36 is greater than

And is less than or equal to

)：

Ser637, Arg638, Arg639, Lys662, Ala664, Ala666, Thr679, Asp680, Ser682, Lys684, Pro707, Gly725, Asn727, Asp748, Pro750, Glu766, Gly769, Pro771, Asn794, Thr808, Asp809, Asp811, Thr812, Leu814, Leu832, Pro835, Asp849, Arg852, His872, Leu873, Val876 and Asp 878.

The structure of the Lrp6E3E4 VHH36 complex also allows identification of VThe following residues on HH36 are core interaction sites (the interatomic distance between Lrp6E3E4 and VHH36 is less than or equal to

)：

Gln1 (modified to pyroglutamate), Val2, Lys3, Ala24, Ser25, Gly26, Arg27, Ile28, Ser30, Ile31, Tyr32, Trp53, Asn73, Asn76, Arg98 and Tyr 100.

The structure of the Lrp6E3E4 VHH36 complex also allows identification of the following residues on VHH36 as core interaction sites (the interatomic distance between Lrp6E3E4 and VHH36 is greater than that of VHH36

And is less than or equal to

)：

Leu4, Ala23, Phe29, Asp33, Arg52, Ser54, Lys71, Asp72, Ala74, Lys75, Ser97 and Gly 99.

Table 3 summary of binding properties of antigen binding proteins of LRP determined by co-crystal structure. In the case of multispecific binders, in addition to the structurally determined epitope information on the LRPs for which the crystal structure is determined, the corresponding residues of the relevant LRP5 are listed using the amino acid single letter code.

These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

Sequence listing

<110> Surrozen Co., Ltd (Surrozen, Inc.)

Li Yang (Li, Yang)

Yuan Zhi Ye (Yuan, Tom Zhiye)

Arsen, Ken, Zong Teng (Sato, Aaron Ken)

Gem leaf Wen (Yeh, Wen-Chen)

Patasaradi-Morpa Cuoma (Sampathkumar, Partha)

<120> anti-LRP 5/6 antibodies and methods of use

<130>SRZN-005/02WO 328202-2032

<150>US 62/607,879

<151>2017-12-19

<150>US 62/680,515

<151>2018-06-04

<160>621

<170> PatentIn version 3.5

<210>1

<211>124

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Val Ile Gly Arg Ser Gly Gly Ile Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Ile Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Thr Arg Arg Pro Phe Asn Ser Tyr Asn Thr Glu Gln Ser Tyr Asp

100 105 110

Ser Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>2

<211>121

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>2

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Gly Val Ser Gly Ser Ile Phe Ser Ile Tyr

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Val Ile Thr Ser Gly Gly Lys Thr Val Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Thr Glu Asp Ala Ala Lys Asn Thr Val Tyr Leu

65 70 7580

Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Asp Ser Arg Ser Ser Trp Tyr Asp Glu Tyr Leu Glu His Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>3

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>3

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Gly Arg Thr Phe Ser Val Tyr

20 25 30

Gly Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val

35 40 45

Ala Ala Val Ser Ala Ser Gly Gly Tyr Thr Trp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Ala Phe

65 70 7580

Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Lys Ala Ala Pro Arg Trp Gly Gly Ala Thr Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210>4

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>4

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Ser Ile Phe Ser Asp Gly

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Val Ile Ser Gly Gly Arg Thr Gly Tyr Ala Asp Phe Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Asn Leu Gln

65 70 75 80

Met Asn Arg Leu Glu Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn Thr

85 90 95

Tyr Pro Phe Pro Ile Tyr Lys Lys Gly Tyr Pro Phe Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210>5

<211>124

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>5

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Val Ile Gly Arg Ser Gly Gly Ile Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Ile Asn Thr Val Tyr

65 70 75 80

Leu GlnMet Asn Asn Leu Thr Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Thr Arg Arg Pro Phe Asn Ser Tyr Asn Thr Glu Gln Ser Tyr Asp

100 105 110

Ser Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>6

<211>121

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>6

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Val Ile Thr Ser Gly Gly Lys Thr Val Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Thr Glu Asp Ala Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Asp Ser Arg Ser Ser Trp Tyr Asp Glu Tyr Leu Glu His Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>7

<211>122

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>7

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ala Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Gly Gly Ile Arg Trp Ser Gly Gly Thr Thr Leu Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Tyr Ala Arg Thr Val Ile Gly Gly Phe Gly Ala Phe Arg Ala His Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>8

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>8

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Met Ile Asn

20 25 30

Thr Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Arg Pro Val Val Ser Glu Thr Thr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Thr Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>9

<211>122

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>9

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Thr Ser Ala Thr Ile Val Ser Ile Tyr

20 25 30

Arg Ile Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val

35 40 45

Ala Gly Ile Thr Ser Ser Gly Arg Thr Ile Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu LysPro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Ser Thr Val Thr Ala Trp Pro Tyr Tyr Gly Pro Asp Tyr Trp

100 105 110

Gly Lys Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>10

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>10

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210>11

<211>124

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>11

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Thr Leu Ser Cys Thr Met Ser Gly Arg Ser Leu Ser Ser Phe

20 25 30

Ala Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ile Ser Arg Gly Asp Gly Tyr Thr Asp Glu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Val Gly Pro Glu Asp Thr Ala Val Tyr His Cys

85 90 95

Ala Ala Val Gln Ala Val Ile Gly Gly Thr Leu Thr Thr Ala Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>12

<211>124

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Arg Tyr

20 25 30

His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Ala Ile Thr Trp Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys ProGlu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Thr Trp Ala Pro Thr Pro Thr Asn Arg Arg Ser Asp Tyr Ala

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>13

<211>119

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Arg Pro Val Val Ser Glu Thr Thr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Ser Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Thr Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>14

<211>126

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>14

Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Ile Ala Asp Asn Arg Tyr Tyr Ala Asp Val Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ala Gly Arg Ser Arg Tyr Leu Tyr Gly Ser Ser Leu Asn Gly Pro

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>15

<211>119

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>15

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Arg Pro Val Val Ser Glu Thr Thr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Ser Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Thr Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>16

<211>121

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>16

Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Phe Ser Met Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ser Ile Arg Trp Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Tyr Ala Asn Ile Tyr Tyr Thr Arg Arg Ala Pro Glu Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>17

<211>119

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Arg Asn Ile Phe Pro Ile Asp

20 25 30

Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Val Thr Ser Gly Gly Arg Ile Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Arg Arg Thr Ile Asp Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Asp Arg Thr Leu Tyr Gly Lys Tyr Lys Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>18

<211>111

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>18

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Ile Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Gly Ile Arg Trp Ser Gly Gly Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Ile Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly

85 90 95

Ser Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210>19

<211>119

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>19

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Arg Pro Val Val Ser Glu Thr Thr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Ser Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Thr Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>20

<211>121

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>20

Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Phe Ser Met Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ser Ile Arg Trp Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Tyr Ala Asn Ile Tyr Tyr Thr Arg Arg Ala Pro Glu Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>21

<211>126

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Asn Thr Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ser Val Ser Trp Arg Tyr Asp Arg Thr Tyr Tyr Thr Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Asp Thr Asn Trp Arg Ala Gly Pro Arg Val Gly Ile Asp Glu

100 105 110

Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210>22

<211>119

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Arg Asn Ile Phe Pro Ile Asp

20 25 30

Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Val Thr Ser Gly Gly Arg Ile Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Arg Arg Thr Ile Asp Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Asp Arg Thr Leu Tyr Gly Lys Tyr Lys Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>23

<211>111

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>23

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Ile Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Gly Ile Arg Trp Ser Gly Gly Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Ile Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly

85 90 95

Ser Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100105 110

<210>24

<211>117

<212>PRT

<213> Artificial Sequence (Sequence)

<220>

<223> preparation-Synthesis of variable heavy chain in laboratory

<400>24

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Ala Asn Ile Asn Ser Ile Glu

20 25 30

Thr Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Ile

35 40 45

Ala Asn Met Arg Gly Gly Gly Tyr Met Lys Tyr Ala Gly Ser Leu Lys

50 55 60

Gly Arg Phe Thr Met Ser Thr Glu Ser Ala Lys Asn Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Val Lys Leu Arg Asp Asp Asp Tyr Val Tyr Arg Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210>25

<211>10

<212>PRT

<213>

<220>

<223> heavy chain CDR1

<400>25

Gly Phe Thr Phe Ser His Tyr Thr Leu Ser

1 5 10

<210>26

<211>17

<212>PRT

<213> Artificial (Artificial)

<220>

<223> heavy chain CDR2

<400>26

Val Ile Ser Gly Asp Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>27

<211>9

<212>PRT

<213> Artificial (Artificial)

<220>

<223> heavy chain CDR3

<400>27

Asn Phe Ile Lys Tyr Val Phe Ala Asn

1 5

<210>28

<211>11

<212>PRT

<213> Artificial (Artificial)

<220>

<223> light chain CDR1

<400>28

Ser Gly Asp Lys Leu Gly Lys Lys Tyr Ala Ser

1 5 10

<210>29

<211>8

<212>PRT

<213> Artificial (Artificial)

<220>

<223>Light chain CDR2

<400>29

Glu Lys Asp Asn Arg Pro Ser Gly

1 5

<210>30

<211>9

<212>PRT

<213> Artificial (Artificial)

<220>

<223>Light chain CDR3

<400>30

Ser Ser Phe Ala Gly Asn Ser Leu Glu

1 5

<210>31

<211>11

<212>PRT

<213> Artificial (Artificial)

<220>

<223> light chain CDR1

<400>31

Ser Gly Asp Asn Ile Gly Ser Phe Tyr Val His

1 510

<210>32

<211>8

<212>PRT

<213> Artificial (Artificial)

<220>

<223> light chain CDR2

<400>32

Asp Lys Ser Asn Arg Pro Ser Gly

1 5

<210>33

<211>9

<212>PRT

<213> Artificial (Artificial)

<220>

<223> light chain CDR3

<400>33

Gln Ser Tyr Ala Asn Thr Leu Ser Leu

1 5

<210>34

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>34

Asp Thr Phe Ala Asn Tyr Gly Phe Ser

1 5

<210>35

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>35

Phe Ala Phe Lys Asp Tyr Tyr Met Thr

1 5

<210>36

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>36

Phe Ala Phe Ser Thr Thr Ala Met Ser

1 5

<210>37

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>37

Phe Ala Leu Ser Gly Tyr Tyr Met Ser

1 5

<210>38

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>38

Phe Pro Phe Arg Tyr Tyr Gly Met Ser

1 5

<210>39

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>39

Phe Pro Phe Glx Tyr Tyr Ser Met Asn

1 5

<210>40

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>40

Phe Arg Phe Ser Ile Ser Thr Met Gly

1 5

<210>41

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>41

Phe Ser Phe Asp Asp Tyr Gly Met Ser

1 5

<210>42

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>42

Phe Ser Phe Glu Thr Tyr Gly Met Ser

1 5

<210>43

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>43

Phe Ser Phe Asn Thr Phe Gly Ile His

1 5

<210>44

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>44

Phe Ser Phe Asn Thr Tyr Ala Met Asn

1 5

<210>45

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>45

Phe Ser Phe Ser Asp Tyr Tyr Met Ser

1 5

<210>46

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>46

Phe Ser Phe Ser Ser Tyr Ser Met Asn

1 5

<210>47

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>47

Phe Thr Phe Asp Ala Tyr Ala Met His

1 5

<210>48

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>48

Phe Thr Phe Asp Asp Tyr Gly Met Ser

1 5

<210>49

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>49

Phe Thr Phe Asp Glu Tyr Ala Met His

1 5

<210>50

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>50

Phe Thr Phe Asn Ser Tyr Ser Met Asp

1 5

<210>51

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>51

Phe Thr Phe Arg Asn Tyr Ala Ile His

1 5

<210>52

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>52

Phe Thr Phe Ser Ala His Gly Met His

1 5

<210>53

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>53

Phe Thr Phe Ser Asp Phe Ala Met Thr

1 5

<210>54

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>54

Phe Thr Phe Ser Asp His Tyr Met Ser

1 5

<210>55

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>55

Phe Thr Phe Ser Asp Tyr Tyr Met Ser

1 5

<210>56

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>56

Phe Thr Phe Ser Lys Thr Asp Met His

1 5

<210>57

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>57

Phe Thr Phe Ser Asn Ser Asp Met Asn

1 5

<210>58

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>58

Phe Thr Phe Ser Asn Tyr Trp Ile His

1 5

<210>59

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>59

Phe Thr Phe Ser Arg His Ser Met Asn

1 5

<210>60

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>60

Phe Thr Phe Ser Ser His Ser Thr His

1 5

<210>61

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>61

Phe Thr Phe Ser Ser Ser Ala Met His

1 5

<210>62

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>62

Phe Thr Phe Ser Ser Ser Trp Met His

1 5

<210>63

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>63

Phe Thr Phe Ser Ser Tyr Ala Met His

1 5

<210>64

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>64

Phe Thr Phe Ser Ser Tyr Ala Met Ser

1 5

<210>65

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>65

Phe Thr Phe Ser Ser Tyr Asp Met His

1 5

<210>66

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>66

Phe Thr Phe Ser Ser Tyr Ser Met Asn

1 5

<210>67

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>67

Phe Thr Phe Ser Ser Tyr Trp Met His

1 5

<210>68

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>68

Phe Thr Phe Ser Ser Glx Glx Met His

1 5

<210>69

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>69

Phe Thr Phe Ser Thr Phe Gly Met His

1 5

<210>70

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>70

Phe Thr Phe Ser Thr His Ala Phe His

1 5

<210>71

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>71

Phe Thr Phe Ser Thr Tyr Gly Met His

1 5

<210>72

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>72

Phe Thr Phe Ser Thr Tyr Ser Met Asn

1 5

<210>73

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>73

Phe Thr Phe Ser Thr Tyr Tyr Met Ser

1 5

<210>74

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>74

Phe Thr Phe Thr Asp Tyr Gly Leu His

1 5

<210>75

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>75

Phe Thr Phe Thr Ser Ser Ala Met Gln

1 5

<210>76

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>76

Phe Thr Phe Thr Ser Ser Ala Val Gln

1 5

<210>77

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>77

Phe Thr Phe Glx Asn Ala Trp Met Ser

1 5

<210>78

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>78

Phe Thr Leu Arg Asn His Trp Leu Ser

1 5

<210>79

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>79

Phe Thr Val Ser Ser Asn Tyr Met Ser

1 5

<210>80

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>80

Gly Asp Leu Ser Ile Tyr Thr Ile Asn

1 5

<210>81

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>81

Gly Ser Phe Ser Asp Tyr Tyr Trp Ser

1 5

<210>82

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>82

Gly Ser Phe Ser Gly Tyr Tyr Trp Thr

1 5

<210>83

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>83

Gly Ser Leu Ser Gly Tyr Tyr Trp Ser

1 5

<210>84

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>84

Gly Thr Phe His Thr Tyr Gly Leu Ser

1 5

<210>85

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>85

Gly Thr Phe Asn Ser Asn Ala Ile Ser

1 5

<210>86

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>86

Gly Thr Phe Asn Thr His Thr Ile Thr

1 5

<210>87

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>87

Gly Thr Phe Ser His Tyr Thr Ile Ser

1 5

<210>88

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>88

Gly Thr Phe Ser Arg Tyr His Met Gly

1 5

<210>89

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>89

Gly Thr Phe Ser Ser His Ala Ile Asn

1 5

<210>90

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>90

Gly Thr Phe Ser Ser Tyr Ala Ile Ser

1 5

<210>91

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>91

Gly Thr Phe Ser Ser Tyr Thr Ile Ser

1 5

<210>92

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>92

Gly Thr Phe Ser Glx Tyr Ala Ile Ser

1 5

<210>93

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>93

Gly Thr Ile Ser Asp Tyr Thr Val Ser

1 5

<210>94

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>94

His Thr Phe Leu Thr Tyr Asp Ile Asn

1 5

<210>95

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>95

His Thr Phe Ser Ser Tyr Ala Met Gly

1 5

<210>96

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>96

Ile Ala Phe Arg Tyr Tyr Asp Met Gly

1 5

<210>97

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>97

Ile Ser Ser Val Tyr Gly Met Gly

1 5

<210>98

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>98

Leu Pro Phe Ser Arg Tyr Ala Met Ala

1 5

<210>99

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>99

Leu Ser Phe Ser Ser Tyr Ala Met Gly

1 5

<210>100

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>100

Leu Ser Ser Gly Arg Pro Phe Ser Ser Tyr Val Met Gly

1 5 10

<210>101

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>101

Leu Thr Phe Asp Asp His Ala Met His

1 5

<210>102

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>102

Leu Thr Phe Ser Asn Ala Ala Met Ala

1 5

<210>103

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>103

Leu Thr Phe Thr Ser His Gly Met Ser

1 5

<210>104

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>104

Leu Thr Val Ser Ser Asn Tyr Met Ser

1 5

<210>105

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>105

Asn Ile Phe Pro Ile Asp Asp Met Ser

1 5

<210>106

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>106

Asn IleAsn Ser Ile Glu Thr Leu Gly

1 5

<210>107

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>107

Arg Ile Phe Ala Ile Tyr Asp Ile Ala

1 5

<210>108

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>108

Arg Ile Phe Ala Ile Tyr Asp Met Ala

1 5

<210>109

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>109

Arg Ile Phe Ser Ile Tyr Asp Ile Ala

1 5

<210>110

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>110

Arg Ile Phe Ser Ile Tyr Asp Met Gly

1 5

<210>111

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>111

Arg Leu Leu Ser Tyr Tyr Ala Leu Ala

1 5

<210>112

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>112

Arg Arg Phe Thr Thr Tyr Gly Met Gly

1 5

<210>113

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>113

Arg Ser Phe Asn Ser Tyr Thr Thr Thr

1 5

<210>114

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>114

Arg Ser Phe Asn Ser Tyr Val Ile Gly

1 5

<210>115

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>115

Arg Ser Phe Ser Asp Phe Phe Met Gly

1 5

<210>116

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>116

Arg Ser Leu Ser Ser Phe Ala Met Gly

1 5

<210>117

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>117

Arg Ser Val Ser Ile Tyr Pro Met Gly

1 5

<210>118

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>118

Arg Ser Val Ser Ser Tyr Asn Met Gly

1 5

<210>119

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>119

Arg Thr Phe Gly Asn Tyr Asp Met Gly

1 5

<210>120

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>120

Arg Thr Phe Gly Ser Asp Val Met Gly

1 5

<210>121

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>121

Arg Thr Phe Gly Thr Tyr Pro Asn Gly

1 5

<210>122

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>122

Arg Thr Phe Asn Ile Asp Asp Met Gly

1 5

<210>123

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>123

Arg Thr Phe Asn Ser Gly Thr Met Gly

1 5

<210>124

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>124

Arg Thr Phe Asn Thr Tyr Ala Met Gly

1 5

<210>125

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>125

Arg Thr Phe Arg Arg Tyr Ala Met Gly

1 5

<210>126

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>126

Arg Thr Phe Arg Ser Tyr Thr Met Gly

1 5

<210>127

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>127

Arg Thr Phe Ser Asp Tyr Ser Met Ala

1 5

<210>128

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>128

Arg Thr Phe Ser Asp Tyr Ser Met Gly

1 5

<210>129

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>129

Arg Thr Phe Ser Met Tyr Asp Met Gly

1 5

<210>130

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>130

Arg Thr Phe Ser Asn Tyr Ala Val Gly

1 5

<210>131

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>131

Arg Thr Phe Ser Arg Tyr Ala Met Ala

1 5

<210>132

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>132

Arg Thr Phe Ser Arg Tyr Ala Met Gly

1 5

<210>133

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>133

Arg Thr Phe Ser Arg Tyr Val Met Gly

1 5

<210>134

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>134

Arg Thr Phe Ser Ser Tyr Ala Met Ala

1 5

<210>135

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>135

Arg Thr Phe Ser Ser Tyr Ala Met Ser

1 5

<210>136

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>136

Arg Thr Phe Ser Ser Tyr Ala Val Gly

1 5

<210>137

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>137

Arg Thr Phe Ser Val Tyr Gly Val Gly

1 5

<210>138

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>138

Arg Thr Phe Ser Tyr Tyr Ala Met Gly

1 5

<210>139

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>139

Arg Thr Leu Ser Ala Tyr Asp Met Gly

1 5

<210>140

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>140

Arg Thr Leu Ser Arg Tyr Ser Met Gly

1 5

<210>141

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>141

Arg Thr Leu Ser Ser Phe Ala Met Gly

1 5

<210>142

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>142

Arg Val Leu Ser Tyr Tyr Ala Met Ala

1 5

<210>143

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>143

Ser Ile Phe Met Ile Asn Thr Met Ala

1 5

<210>144

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>144

Ser Ile Phe Arg Leu Gly Thr Met Tyr

1 5

<210>145

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>145

Ser Ile Phe Ser Asp Gly Ala Met Gly

1 5

<210>146

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>146

Ser Ile Phe Ser Ile Asn Thr Met Gly

1 5

<210>147

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>147

Ser Ile Phe Ser Ile Tyr Ala Met Gly

1 5

<210>148

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>148

Ser Ile Ser Ser Phe Asn Thr Met Gly

1 5

<210>149

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>149

Ser Ile Val Arg Ser Leu Pro Met Ala

1 5

<210>150

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>150

Ser Leu Phe Ser Phe Asn Ala Met Gly

1 5

<210>151

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>151

Ser Leu Phe Ser Phe Asn Ala Val Gly

1 5

<210>152

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>152

Ser Val Phe Thr Thr Phe Ala Lys Gly

1 5

<210>153

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>153

Thr Ile Phe Ser Ile Asn Thr Met Gly

1 5

<210>154

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>154

Thr Ile Val Ser Ile Tyr Arg Ile Asn

1 5

<210>155

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>155

Val Ile Phe Ala Leu Tyr Asp Ile Ala

1 5

<210>156

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>156

Val Thr Phe Ser Arg Tyr Pro Ile Ser

1 5

<210>157

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>157

Tyr Gly Phe Thr Gly Tyr Tyr Ile His

1 5

<210>158

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>158

Tyr Ile Phe Thr Asp Tyr Tyr Met His

1 5

<210>159

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>159

Tyr Ser Phe Thr Arg Thr Asp Met His

1 5

<210>160

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>160

Tyr Thr Phe Ala Ser Tyr Asp Ile His

1 5

<210>161

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>161

Tyr Thr Phe Ser Tyr Arg Tyr Leu His

1 5

<210>162

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>162

Tyr Thr Phe Thr Asp Tyr Phe Met Asn

1 5

<210>163

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>163

Tyr Thr Phe Thr Gly Tyr Tyr Ile His

1 5

<210>164

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>164

Tyr Thr Phe Thr Gly Tyr Tyr Met His

1 5

<210>165

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>165

Tyr Thr Phe Thr Asn Asn Phe Met His

1 5

<210>166

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>166

Tyr Thr Phe Thr Asn Tyr Cys Met His

1 5

<210>167

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>167

Tyr Thr Phe ThrSer Tyr Ala Met Asn

1 5

<210>168

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>168

Tyr Thr Phe Thr Ser Tyr Asp Ile Asn

1 5

<210>169

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>169

Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5

<210>170

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>170

Tyr Thr Phe Thr Tyr Arg Tyr Leu His

1 5

<210>171

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>171

Tyr Thr Ile Ser Asn Tyr Tyr Ile His

1 5

<210>172

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR1

<400>172

Tyr Glx Phe Thr Asp Tyr Tyr Met His

1 5

<210>173

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>173

Ala Ala Ile Lys Trp Ser Gly Thr Asn Thr Tyr Tyr Ala

1 5 10

<210>174

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>174

Ala Ala Ile Asn Trp Ser Gly Asp Ser Thr Lys Tyr Ala

1 5 10

<210>175

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>175

Ala Ala Ile Gln Trp Ser Ala Asp Asn Thr Phe Tyr Ala

1 5 10

<210>176

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>176

Ala Ala Ile Arg Trp Ser Gly Asp Asn Thr Tyr Tyr Ala

1 5 10

<210>177

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>177

Ala Ala Ile Arg Trp Ser Gly Gly Gly Thr Tyr Tyr Ala

1 5 10

<210>178

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>178

Ala Ala Ile Ser Gly Ser Gly Gly Ser Thr Thr Tyr Ala

1 5 10

<210>179

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>179

Ala Ala Ile Ser Gln Ser Gly Tyr Val Arg Tyr Tyr Ala

1 5 10

<210>180

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>180

Ala Ala Ile Ser Arg Phe Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>181

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>181

Ala Ala Ile Ser Arg Phe Gly Gly Ser Thr Tyr Tyr Val

1 5 10

<210>182

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>182

Ala Ala Ile Ser Arg Asn Gly Asp Lys Ser His Tyr Ser

1 5 10

<210>183

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>183

Ala Ala Ile Ser Arg Arg Gly Gly Ile Ile Glu Tyr Gly

1 5 10

<210>184

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>184

Ala Ala Ile Ser Arg Ser Gly Ala Asn Thr Ala Tyr Ser

1 5 10

<210>185

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>185

Ala Ala Ile Ser Arg Ser Gly Asp Arg Ile Tyr Tyr Ser

1 5 10

<210>186

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>186

Ala Ala Ile Ser Arg Ser Gly Gly Ile Tyr Tyr Ala

1 5 10

<210>187

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>187

Ala Ala Ile Ser Arg Ser Gly Val Ser Thr Tyr Tyr Ala

1 5 10

<210>188

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>188

Ala Ala Ile Ser Trp Gly Gly Arg Thr Ala Tyr Ala

1 5 10

<210>189

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>189

Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Lys Tyr Ala

1 5 10

<210>190

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>190

Ala Ala Ile Ser Tyr Ser Gly Gly Ser Thr Lys Tyr Ala

1 5 10

<210>191

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>191

Ala Ala Ile Thr Arg Ser Gly Ser Asn Thr Tyr Tyr Ala

1 5 10

<210>192

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>192

Ala Ala Ile Thr Trp Asn Gly Arg Ser Ser Asp Tyr Ala

1 5 10

<210>193

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>193

Ala Ala Ile Thr Trp Arg Gly Gly Ile Thr Tyr Tyr Ala

1 5 10

<210>194

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>194

Ala Ala Ile Thr Trp Arg Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>195

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>195

Ala Ala Val Ser Ala Ser Gly Gly Tyr Thr Trp Tyr Ala

1 5 10

<210>196

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>196

Ala Ala Val Thr Trp Arg Ser Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>197

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>197

Ala Gly Ile Arg Trp Ser Gly Ser Thr Leu Tyr Ala

1 5 10

<210>198

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>198

Ala Gly Ile Ser Trp Ile Ala Asp Asn Arg Tyr Tyr Ala

1 5 10

<210>199

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>199

Ala Gly Ile Thr Arg Gly Gly Ala Thr Thr Tyr Tyr Ser

1 5 10

<210>200

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>200

Ala Gly Ile Thr Ser Ser Gly Arg Thr Ile Tyr Ala

1 5 10

<210>201

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>201

Ala Gly Met Ser Gly Glu Gly Arg Asn Thr Lys Tyr Arg

1 5 10

<210>202

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>202

Ala Leu Thr Gly Trp Gly Asp Gly Ser Thr Thr Tyr Tyr Glu

1 5 10

<210>203

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>203

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala

1 5 10

<210>204

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>204

Ala Asn Met Arg Gly Gly Gly Tyr Met Lys Tyr Ala

1 5 10

<210>205

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>205

Ala Arg Ile Gly Trp Asn Gly Gly Ser Ile Val Tyr Ala

1 5 10

<210>206

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>206

Ala Arg Ile Ser Arg Gly Asp Gly Tyr Thr Asp Glu Ala

1 5 10

<210>207

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>207

Ala Ser Ile Gly Lys Ser Gly Ser Thr Asn Tyr Ala

1 5 10

<210>208

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>208

Ala Ser Ile Arg Trp Ser Gly Gln Ser Pro Tyr Tyr Ala

1 5 10

<210>209

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>209

Ala Ser Ile Arg Trp Ser Ser Gly Asn Thr Trp Tyr Ala

1 5 10

<210>210

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>210

Ala Ser Ile Ser Ser Gly Gly Arg Thr Asn Tyr Ala

1 5 10

<210>211

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>211

Ala Ser Ile Ser Ser Gly Ser Arg Thr Asn Tyr Ala

1 5 10

<210>212

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>212

Ala Ser Ile Ser Ser Thr Ser Gly Ser Lys Tyr Tyr Ala

1 5 10

<210>213

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>213

Ala Ser Ile Thr Ala Ser Ser Asp Arg Thr Phe Tyr Ala

1 5 10

<210>214

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>214

Ala Ser Val Ser Trp Arg Tyr Asp Arg Thr Tyr Tyr Thr

1 5 10

<210>215

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>215

Ala Thr Ile Asn Asp Ala Gln Arg Tyr Tyr Ala

1 5 10

<210>216

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>216

Ala Thr Ile Arg Pro Val Val Ser Glu Thr Thr Tyr Ala

1 5 10

<210>217

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>217

Ala Thr Ile Arg Pro Val Val Thr Glu Thr Asp Tyr Ala

1 5 10

<210>218

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>218

Ala Thr Ile Arg Pro Val Val Thr Gln Ile Asp Tyr Ala

1 5 10

<210>219

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>219

Ala Thr Ile Ser Ala Ser Gly Gly Asn Thr Ala Tyr Ala

1 5 10

<210>220

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>220

Ala Thr Ile Ser Arg Ser Gly Gly Asn Thr Tyr Tyr Ala

1 5 10

<210>221

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>221

Ala Thr Ile Ser Trp Ser Gly Ser Ser Ala Asn Tyr Glu

1 5 10

<210>222

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>222

Ala Thr Met Thr Ser Gly Gly Asn Thr Asn Tyr Ala

1 5 10

<210>223

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>223

Ala Thr Val Thr Ser Gly Gly Arg Ile Asn Tyr Ala

1 5 10

<210>224

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>224

Ala Val Ile Gly Arg Ser Gly Gly Ile Lys Tyr Tyr Ala

1 5 10

<210>225

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>225

Ala Val Ile Ser Gly Gly Arg Thr Gly Tyr Ala

1 5 10

<210>226

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>226

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

1 5 10

<210>227

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>227

Ala Val Ile Thr Ser Gly Gly Lys Thr Val Tyr Ala

1 5 10

<210>228

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>228

Ala Val Ile Thr Thr Gly Gly Asp Thr Ser TyrSer

1 5 10

<210>229

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>229

Ala Val Thr Ser Tyr Asp Gly Gly Lys Lys Asn Tyr Ala

1 5 10

<210>230

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>230

Ala Tyr Ile Thr Gly Gly Gly Arg Thr Met Asp Gly

1 5 10

<210>231

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>231

Gly Ala Ile Ser Arg Ser Gly Asn Asn Thr Tyr Tyr Ala

1 5 10

<210>232

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>232

Gly Glu Ile Asn His Ser Gly Ala Thr Asn Tyr Asn

1 5 10

<210>233

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>233

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

1 5 10

<210>234

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>234

Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala

1 5 10

<210>235

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>235

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asp Tyr Ala

1 5 10

<210>236

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>236

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala

1 5 10

<210>237

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>237

Gly Gly Ile Ile Pro Leu Phe Gly Thr Ala Asn Tyr Ala

1 5 10

<210>238

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>238

Gly Gly Ile Ile Pro Val Phe Gly Thr Ala Asp Tyr Ala

1 5 10

<210>239

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>239

Gly Gly Ile Ile Pro Val Phe Gly Thr Ala Asn Tyr Ala

1 5 10

<210>240

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>240

Gly Gly Ile Arg Trp Ser Gly Gly Thr Thr Leu Tyr Pro

1 5 10

<210>241

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>241

Gly Gly Ile Val Pro Ala Tyr Arg Arg Ala Asn Tyr Ala

1 5 10

<210>242

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>242

Gly Gly Val Ile Pro Ile Phe Gly Thr Ala Asp Tyr Ala

1 5 10

<210>243

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>243

Gly His Val Asp Pro Gly Asp Gly Glu Thr Ile Tyr Ala

1 5 10

<210>244

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>244

Gly Ile Ile Asn Pro Ser AspGly Ser Thr Ser His Ala

1 5 10

<210>245

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>245

Gly Ile Ile Asn Pro Ser Gly Asp Ser Thr Arg Phe Ala

1 5 10

<210>246

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>246

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ile Tyr Ala

1 5 10

<210>247

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>247

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Asn Tyr Ala

1 5 10

<210>248

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>248

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala

1 5 10

<210>249

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>249

Gly Met Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala

1 5 10

<210>250

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>250

Gly Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala

1 5 10

<210>251

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>251

Gly Arg Ile Ile Pro Ile His Gly Ile Ala Asn Tyr Ala

1 5 10

<210>252

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>252

Gly Arg Ile Ile Pro Ile Leu Gly Arg Ala Asn Tyr Ala

1 5 10

<210>253

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>253

Gly Arg Ile Ile Pro Ile Leu Gly Arg Thr Asn Tyr Ala

1 5 10

<210>254

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>254

Gly Arg Ile Ile Pro Ile Leu Gly Ser Thr Asn Tyr Ala

1 5 10

<210>255

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>255

Gly Arg Ile Ile Pro Val Leu Lys Ile Thr Asn Tyr Ala

1 5 10

<210>256

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>256

Gly Arg Ile Asn Pro Asn Gly Gly Gly Thr Ile Tyr Ala

1 5 10

<210>257

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>257

Gly Arg Ile Thr Pro Arg Leu Gly Ile Ala Asn Tyr Ala

1 5 10

<210>258

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>258

Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser

1 5 10

<210>259

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>259

Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Thr Tyr Ala

1 5 10

<210>260

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>260

Gly Trp Ile Asn Pro Asn Ser Gly Asp Thr Asn Tyr Ala

1 5 10

<210>261

<400>261

000

<210>262

<400>262

000

<210>263

<400>263

000

<210>264

<400>264

000

<210>265

<400>265

000

<210>266

<400>266

000

<210>267

<400>267

000

<210>268

<400>268

000

<210>269

<400>269

000

<210>270

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>270

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala

1 5 10

<210>271

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>271

Gly Trp Ile Ser Ala Asn Asn Gly Asn Thr Asp Tyr Ala

1 5 10

<210>272

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>272

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala

1 5 10

<210>273

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa can be any naturally occurring amino acid

<400>273

Gly Xaa Val Asn Ala Gly Asn Gly Asn Thr Thr Tyr Ala

1 5 10

<210>274

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>274

Gly Tyr Ile Ser Ala Tyr Thr Gly His Thr Ser Tyr Ala

1 5 10

<210>275

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>275

Ser Ala Ile Gly Ala Gly Gly Gly Thr Tyr Tyr Ala

1 5 10

<210>276

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>276

Ser Ala Ile Gly Thr Gly Gly Asp Thr Tyr Tyr Ala

1 5 10

<210>277

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>277

Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala

1 5 10

<210>278

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>278

Ser Ala Ile Arg Gly Ser Gly Glu Arg Thr Tyr Tyr Ala

1 5 10

<210>279

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>279

Ser Ala Ile Ser Gly Arg Asp Gly Arg ThrTyr Tyr Ala

1 5 10

<210>280

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>280

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>281

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>281

Ser Ala Ile Ser Ser Gly Ser Asp Arg Thr Tyr Tyr Ala

1 5 10

<210>282

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>282

Ser Ala Ile Thr Trp Ser Gly Gly Arg Thr Tyr Tyr Ala

1 5 10

<210>283

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>283

Ser Gly Ile Arg Trp Ser Gly Gly Thr Ser Tyr Ala

1 5 10

<210>284

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>284

Ser Gly Ile Ser Glu Ser Gly Gly Arg Thr Tyr Tyr Ala

1 5 10

<210>285

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>285

Ser Gly Ile Ser Gly Ser Gly Gly Arg Thr His Tyr Ala

1 5 10

<210>286

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>286

Ser Gly Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>287

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>287

Ser Gly Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val

1 5 10

<210>288

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>288

Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Ala

1 5 10

<210>289

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>289

Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala

1 5 10

<210>290

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>290

Ser Gly Ile Ser Trp Asn Ser Gly Thr Thr Gly Tyr Ser

1 5 10

<210>291

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>291

Ser Gly Ile Thr Ser Asn Gly Gly Ala Thr Tyr Tyr Ala

1 5 10

<210>292

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>292

Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Ala

1 5 10

<210>293

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>293

Ser Met Ile Ser Tyr Asn Gly Gly Arg Ala Phe Tyr Ala

1 5 10

<210>294

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>294

Ser Ser Ile Ser Gly Ser Gly Gly Val Thr Tyr Tyr Ala

1 5 10

<210>295

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>295

Ser Ser Ile Ser Pro Arg Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>296

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>296

Ser Ser Ile Ser Ser Ser Ser Thr Tyr Ile Arg Tyr Ala

1 5 10

<210>297

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>297

Ser Ser Ile Ser Val Ser Ser Gly Thr Thr His Tyr Ala

1 5 10

<210>298

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>298

Ser Thr Ile Asn Pro Gly Gly Leu Ser Lys Ser Tyr Ala

1 5 10

<210>299

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>299

Ser Thr Ile Ser Asp Thr Asn Ser Gly Thr Tyr Tyr Ala

1 5 10

<210>300

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>300

Ser Thr Ile Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala

1 5 10

<210>301

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>301

Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>302

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>302

Ser Thr Ile Thr Ser Ser Gly Gly Ser Thr Tyr Tyr Ala

1 5 10

<210>303

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>303

Ser Thr Ile Thr Thr Asp Ser Arg Gly Thr Tyr Tyr Ala

1 5 10

<210>304

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>304

Ser Thr Leu Ser Gly Asp Ala Asn Asn Ala Tyr Tyr Ala

1 5 10

<210>305

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>305

Ser Val Ile Ser Ser Gly Gly Thr Ile Tyr Tyr Ala

1 5 10

<210>306

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>306

Ser Tyr Ile Ser Gly Asp Ser Gly Tyr Thr Asn Tyr Ala

1 5 10

<210>307

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>307

Ser Tyr Ile Ser Gly Asn Ser Gly Tyr Thr Asn Tyr Ala

1 5 10

<210>308

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>308

Ser Tyr Ile Ser Ser Ser Gly Arg Thr Ile Phe Tyr Ala

1 5 10

<210>309

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>309

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala

1 5 10

<210>310

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>310

Ser Tyr Ser Ser Gly Asn Ser Gly Tyr Thr Asn Tyr Ala

1 5 10

<210>311

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>311

Ser Tyr Val Ser Asp Ser Gly Ser Ser Val Tyr Tyr Ala

1 5 10

<210>312

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR2

<400>312

Glx His Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala

1 5 10

<210>313

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>313

Cys Ala Ala Ala Arg Arg Ser Gly Thr Tyr Asp Ile Gly Gln Tyr Leu

1 5 10 15

Arg Glu Ser Ala Tyr Val Phe Trp

20

<210>314

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>314

Cys Ala Ala Ala Tyr Ser Tyr Ser Gln Tyr Gly Ser Ser Tyr Ser Tyr

1 5 10 15

Trp

<210>315

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>315

Cys Ala Ala Asp Asp Leu Gly Leu Glu Leu His Tyr Trp

1 5 10

<210>316

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>316

Cys Ala Ala Asp Pro Arg Gly Val Thr Leu Pro Arg Ala Thr Ala Tyr

1 5 10 15

Glu Tyr Trp

<210>317

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>317

Cys Ala Ala Asp Arg Ile Glu Asn Tyr Leu Gly Arg Tyr Tyr Asp Pro

1 5 10 15

Ser Glu Tyr Glu Tyr Trp

20

<210>318

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>318

Cys Ala Ala Asp Thr Asn Trp Arg Ala Gly Pro Arg Val Gly Ile Asp

1 5 10 15

Glu Tyr Ala Tyr Trp

20

<210>319

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>319

Cys Ala Ala Gly Pro Asn Trp Ser Thr Arg Asn Arg Glu Tyr Asp Tyr

1 5 10 15

Trp

<210>320

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>320

Cys Ala Ala Gly Ser Thr Val Val Ala Glu Phe Asn Tyr Trp

1 5 10

<210>321

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>321

Cys Ala Ala Lys Phe Gly Val Leu Ala Thr Thr Glu Ser Arg His Asp

1 5 10 15

Tyr Trp

<210>322

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>322

Cys Ala Ala Gln Phe Arg Asn Asp Tyr Gly Leu Arg Tyr Gln Ser Thr

1 5 10 15

Asn Asn Tyr Trp

20

<210>323

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>323

Cys Ala Ala Arg Thr Ser Gly Gly Leu Phe His Tyr Arg Arg Ser Asp

1 5 10 15

His Trp Asp Thr Trp

20

<210>324

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>324

Cys Ala Ala Ser Met Glu Ala Met Asn Ser Leu Arg Val Asn Lys Glu

1 5 10 15

Arg Tyr Tyr Gln Ser Trp

20

<210>325

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>325

Cys Ala Ala Ser Val Tyr Ile Ser Arg Arg Asp Ser Asp Tyr Gly Tyr

1 5 10 15

Trp

<210>326

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>326

Cys Ala Ala Val Phe Thr Gly Arg Phe Tyr Gly Arg Pro Pro Arg Glu

1 5 10 15

Lys Tyr Asp Tyr Trp

20

<210>327

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>327

Cys Ala Ala Val Gln Ala Val Ile Gly Gly Thr Leu Thr Thr Ala Tyr

1 5 10 15

Asp Tyr Trp

<210>328

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>328

Cys Ala Ala Tyr Ser Thr Phe Asn Thr Asp Val Ala Ser Met Lys Pro

1 5 10 15

Asp Tyr Trp

<210>329

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>329

Cys Ala Gly Asp Arg Ser Arg Tyr Leu Tyr Gly Asp Ser Leu Arg Gly

1 5 10 15

Pro Tyr Gly Tyr Trp

20

<210>330

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>330

Cys Ala Ile Val Arg Gly Lys Lys Trp Tyr Phe Asp Leu Trp

1 5 10

<210>331

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>331

Cys Ala Lys Ala Gln Ala Thr Gly Trp Ser Gly Tyr Tyr Thr Phe Asp

1 5 10 15

Tyr Trp

<210>332

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>332

Cys Ala Lys Asp Asp Phe Ser Leu Tyr Gly Met Asp Val Trp

1 5 10

<210>333

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>333

Cys Ala Lys Asp Gly Thr Asp Gly Arg Phe Asp Pro Trp

1 5 10

<210>334

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>334

Cys Ala Lys Asp Leu Gly Ile Gln Leu Pro Asp Tyr Tyr Phe Asp Tyr

1 5 10 15

Trp

<210>335

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>335

Cys Ala Lys Asp Leu Gly Arg Ala Ala Ala Gly Ser Met Asp Val Trp

1 5 10 15

<210>336

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>336

Cys Ala Lys Asp Met Val His Leu Ile Val Ala Leu Ala Ile Asp Tyr

1 5 10 15

Trp

<210>337

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>337

Cys Ala Lys Asp Ser Gly Leu Val

1 5

<210>338

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>338

Cys Ala Lys Asp Ser Gly Asn Tyr Gly Tyr Tyr Gly Met Asp Val Trp

1 5 10 15

<210>339

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>339

Cys Ala Lys Glu Asp Tyr Asp Ser Ser Gly Tyr Tyr Tyr Tyr Tyr Phe

1 5 10 15

Gln His Trp

<210>340

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>340

Cys Ala Lys Gly Gly Asp Tyr Phe Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>341

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>341

Cys Ala Lys Gly Gly Asp Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>342

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>342

Cys Ala Lys Gly Gly Arg Asp Gly Tyr Lys Gly Tyr Phe Asp Tyr Trp

1 5 10 15

<210>343

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>343

Cys Ala Lys Gly Gly Ser Leu Asp Met Asp Val Trp

1 5 10

<210>344

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>344

Cys Ala Lys Gly Gly Tyr Glu Leu Asp Tyr Trp

1 5 10

<210>345

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>345

Cys Ala Lys Gly Trp Leu Asp Phe Asp Tyr Trp

1 5 10

<210>346

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>346

Cys Ala Lys Ser Ile Ala Ala Ala Gly Thr Gly Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>347

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>347

Cys Ala Lys Thr Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Thr Phe Asp

1 5 10 15

Tyr Trp

<210>348

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>348

Cys Ala Lys Val Ala Ser Gly Trp Ser Trp Pro Phe Asp Ile Trp

1 5 10 15

<210>349

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>349

Cys Ala Leu Thr Trp Ala Pro Thr Pro Thr Asn Arg Arg Ser Asp Tyr

1 5 10 15

Ala Tyr Trp

<210>350

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>350

Cys Ala Asn Gly Leu Glu Asp Ala Tyr Ala Phe Asp Ile Trp

1 5 10

<210>351

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>351

Cys Ala Pro Ala Leu Thr Asp Ala Gly Ser Phe Asp Tyr Trp

1 5 10

<210>352

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>352

Cys Ala Pro Tyr Tyr Tyr Asp Lys Ser Ala Lys Pro Leu Arg Ser Tyr

1 5 10 15

Phe Asp His Trp

20

<210>353

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>353

Cys Ala Arg Ala Ala Gly Asn Phe Trp Ser Gly Tyr Tyr Thr Phe Asp

1 5 10 15

Tyr Trp

<210>354

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>354

Cys Ala Arg Ala Gly Ile Ala Ala Ala Pro Gly Ser Arg Asn Tyr Tyr

1 5 10 15

Gly Met Asp Val Trp

20

<210>355

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>355

Cys Ala Arg Ala Gly Thr Asn Trp Gly Gly Trp Tyr Phe Asp Leu Trp

1 5 10 15

<210>356

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>356

Cys Ala Arg Ala Gly Tyr Gly Arg Tyr Tyr Tyr Gly Met Asp Val Trp

1 5 10 15

<210>357

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>357

Cys Ala Arg Ala Gly Tyr Trp Ser Gly Tyr Gly Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>358

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>358

Cys Ala Arg Ala Gly Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ala Phe Asp

1 5 10 15

Ile Trp

<210>359

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>359

Cys Ala Arg Ala Thr Gly Ser Gly Trp Tyr Thr Asp Leu Gly Tyr Trp

1 5 10 15

<210>360

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>360

Cys Ala Arg Asp Ala Gly Gly Asp Tyr Asp Tyr Trp

1 5 10

<210>361

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>361

Cys Ala Arg Asp Ala Ser Gly Gly Ser Thr Gly Trp Tyr Tyr Phe Asp

1 5 10 15

Ser Trp

<210>362

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>362

Cys Ala Arg Asp Asp Gly Leu Gly Gly Met Asp Val Trp

1 5 10

<210>363

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>363

Cys Ala Arg Asp Asp Ser Met Gly Ala Phe Asp Ile Trp

1 5 10

<210>364

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>364

Cys Ala Arg Asp Glu Val Glu Gly Gly Met Asp Val Trp

1 5 10

<210>365

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>365

Cys Ala Arg Asp Phe Leu Gly Ser Thr Gly Asp Tyr Trp

1 5 10

<210>366

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>366

Cys Ala Arg Asp Gly Gly Ile Arg Asp Phe Asp Tyr Trp

1 5 10

<210>367

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>367

Cys Ala Arg Asp His Ser Ser Gly Trp Arg His Tyr Phe Asp Tyr Trp

1 5 10 15

<210>368

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>368

Cys Ala Arg Asp Lys Gly Tyr Ala Phe Asp Ile Trp

1 5 10

<210>369

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>369

Cys Ala Arg Asp Leu Asp Tyr Trp

1 5

<210>370

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>370

Cys Ala Arg Asp Leu Gly Gly Thr Ala Asp Tyr Trp

1 5 10

<210>371

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>371

Cys Ala Arg Asp Leu Arg Asn Trp Gly Ser Pro Tyr Trp Tyr Phe Asp

1 5 10 15

Leu Trp

<210>372

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>372

Cys Ala Arg Asp Gln Asp Tyr Gly Asp Tyr Gly Trp Tyr Tyr Tyr Gly

1 5 10 15

Met Asp Val Trp

20

<210>373

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>373

Cys Ala Arg Asp Arg Glu Gln Gln Ile Leu Asp Tyr Trp

1 5 10

<210>374

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>374

Cys Ala Arg Asp Ser Asp Trp Gly Val Val Asp Pro Trp

1 5 10

<210>375

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>375

Cys Ala Arg Asp Val Gly Ala Phe Asp Ile Trp

1 5 10

<210>376

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>376

Cys Ala Arg Asp Trp Glu Leu Tyr Gly Met Asp Val Trp

1 5 10

<210>377

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>377

Cys Ala Arg Asp Trp Gly Ile Ala Ala Ala Gly Asp Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val Trp

20

<210>378

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>378

Cys Ala Arg Asp Tyr Ser Asp Arg Ser Gly Ile Asp Tyr Trp

1 5 10

<210>379

<211>18

<212>PRT

<213> Artificial sequence (artificacial sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>379

Cys Ala Arg Asp Tyr Tyr Gly Ser Gly Ser Tyr Asn Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>380

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>380

Cys Ala Arg Glu Ala Ala Glu Ile Pro Val Gly Ala Phe Asp Ile Trp

1 5 10 15

<210>381

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>381

Cys Ala Arg Glu Ala Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

1 5 10 15

<210>382

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>382

Cys Ala Arg Glu Glu Gly Val Gly Gly Met Asp Val Trp

1 5 10

<210>383

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>383

Cys Ala Arg Glu Glu Arg Gly Ala Thr Gly Arg Ala Phe Asp Ile Trp

1 5 10 15

<210>384

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>384

Cys Ala Arg Glu Gly Gly Tyr Tyr Phe Asp Tyr Trp

1 5 10

<210>385

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>385

Cys Ala Arg Glu Ser Ala Leu Tyr Ser Ser Ser Trp Tyr Tyr Tyr Tyr

1 5 10 15

Tyr Gly Met Asp Val Trp

20

<210>386

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>386

Cys Ala Arg Glu Val Ala Val Lys Asp Tyr Tyr Tyr Tyr Tyr Met Asp

1 5 10 15

Val Trp

<210>387

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>387

Cys Ala Arg Glu Tyr Ser Tyr Gly Tyr Phe Arg Tyr Trp

1 5 10

<210>388

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>388

Cys Ala Arg Gly Asp Leu Glu Phe Asp Tyr Trp

1 5 10

<210>389

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>389

Cys Ala Arg Gly Glu Gln Trp Leu Val Trp Gly Phe Asp Pro Trp

1 5 10 15

<210>390

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>390

Cys Ala Arg Gly Gly Asp Phe Asp Tyr Trp

1 5 10

<210>391

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>391

Cys Ala Arg Gly Gly Asp Ser Ser Gly Tyr Tyr Tyr Tyr Ala Phe Asp

1 5 10 15

Ile Trp

<210>392

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>392

Cys Ala Arg Gly Gly Gly Pro Asn Glu His Asp Tyr Tyr Phe Asp Tyr

1 5 10 15

Trp

<210>393

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>393

Cys Ala Arg Gly Gly Asn Thr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>394

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>394

Cys Ala Arg Gly Gly Ser Gly Ser Tyr Tyr Tyr Ala Phe Asp Ile Trp

1 5 10 15

<210>395

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>395

Cys Ala Arg Gly Gly Ser Gly Trp Ser Asn Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>396

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>396

Cys Ala Arg Gly Gly Tyr Ser Thr Leu Asp Tyr Trp

1 5 10

<210>397

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>397

Cys Ala Arg Gly Leu Tyr Lys Arg Tyr Ser Tyr Gly Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>398

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>398

Cys Ala Arg Gly Asn Leu Asp Phe Asp Tyr Trp

1 5 10

<210>399

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>399

Cys Ala Arg Gly Asn Pro Tyr Tyr Phe Asp Tyr Trp

1 5 10

<210>400

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>400

Cys Ala Arg Gly Pro Ala Ala Ile Gly Ile Leu Gly Trp Phe Asp Pro

1 5 10 15

Trp

<210>401

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>401

Cys Ala Arg Gly Pro Tyr Tyr Phe Asp Tyr Trp

1 5 10

<210>402

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>402

Cys Ala Arg Gly Arg Gly Lys Lys Asn Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>403

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>403

Cys Ala Arg Gly Arg Gly Tyr Ser Tyr Gly Tyr Tyr Ala Phe Asp Ile

1 5 10 15

Trp

<210>404

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>404

Cys Ala Arg Gly Ser Asp Cys Ser Gly Gly Ser Cys Tyr Tyr Ser Phe

1 5 10 15

Asp Tyr Trp

<210>405

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>405

Cys Ala Arg Gly Ser Gly Trp Ser Gly Leu Asp Tyr Trp

1 5 10

<210>406

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>406

Cys Ala Arg Gly Ser Gly Tyr Tyr Gly Pro Gly Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>407

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>407

Cys Ala Arg Gly Ser Tyr Gly Met Asp Val Trp

1 5 10

<210>408

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>408

Cys Ala Arg Gly Thr Thr Gly Lys Gly Tyr Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>409

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>409

Cys Ala Arg Gly Val Ser Ser Gly Tyr Tyr Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>410

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>410

Cys Ala Arg Gly Tyr Gly Asp Tyr Asp Leu Trp

1 5 10

<210>411

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>411

Cys Ala Arg Gly Tyr Tyr Asp Phe Asp Tyr Trp

1 5 10

<210>412

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>412

Cys Ala Arg His Leu Ser Ser Gly Tyr Leu Ser Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210>413

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>413

Cys Ala Arg His Pro Gly Ser Phe Gly Gly Tyr Ser Tyr Ala Trp Tyr

1 5 10 15

Tyr Tyr Tyr Gly Met Asp Val Trp

20

<210>414

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>414

Cys Ala Arg Leu Asp Tyr Gly Glu Thr Glu Gly Asn Gly Asp Trp

1 5 10 15

<210>415

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>415

Cys Ala Arg Leu Gly Ser Tyr Gly Ser Pro Tyr Tyr Tyr Tyr Gly Met

1 5 10 15

Asp Val Trp

<210>416

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>416

Cys Ala Arg Leu Gly Ser Tyr Pro Gly Pro Tyr Tyr Tyr Tyr Met Asp

1 5 10 15

Val Trp

<210>417

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>417

Cys Ala Arg Arg Gly Gly Asp Val Thr Val Pro Ala Ala Tyr Tyr Ala

1 5 1015

Met Asp Val Trp

20

<210>418

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>418

Cys Ala Arg Thr Leu Ser Gly Tyr Ser Ser Ser Trp Tyr Val Phe Asp

1 5 10 15

Tyr Trp

<210>419

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>419

Cys Ala Arg Tyr Ser Gly Tyr Asp Phe Asp Tyr Trp

1 5 10

<210>420

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>420

Cys Ala Ser Ala Ala Asp Phe Asp Tyr Trp

1 5 10

<210>421

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>421

Cys Ala Ser Ala Lys Asn Asp Phe Trp Ser Gly Tyr Phe Ala Phe Asp

1 5 10 15

Tyr Trp

<210>422

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>422

Cys Ala Ser Asp Ile Val Val Asp Asp Ala Phe Asp Thr Trp

1 5 10

<210>423

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>423

Cys Ala Ser Gly Asp Thr Tyr Asp Leu Tyr Ser Leu Asp Val Trp

15 10 15

<210>424

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>424

Cys Ala Ser Gly Ser Tyr Tyr Ser Asp Phe Asp Tyr Trp

1 5 10

<210>425

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>425

Cys Ala Ser Gly Tyr Ser Tyr Gly Leu Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

Trp

<210>426

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>426

Cys Ala Ser Ser Val Val Pro Ala Gly Pro Ala Gly Val Tyr Ala Phe

15 10 15

Asp Ile Trp

<210>427

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>427

Cys Ala Ser Thr Val Thr Thr Asp Ala Phe Asp Ile Trp

1 5 10

<210>428

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>428

Cys Ala Ser Tyr Phe Gly Val Met Asp Val Trp

1 5 10

<210>429

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>429

Cys Ala Thr Ala Tyr Gly Ser Ser Ser Leu Asn Ile Asp Tyr Trp

1 5 10 15

<210>430

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>430

Cys Ala Thr Asp Glu Tyr Ser Ser Ser Tyr Ala Phe Asp Ile Trp

1 5 10 15

<210>431

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>431

Cys Ala Thr Asp Tyr Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp

1 5 10 15

<210>432

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>432

Cys Ala Thr Glu Ala Ala Leu Asp Ala Phe Asp Ile Trp

1 5 10

<210>433

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>433

Cys Ala Thr Gly Pro Asn Ser Ile Tyr

1 5

<210>434

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>434

Cys Ala Thr Arg Arg Pro Phe Asn Ser Tyr Asn Thr Glu Gln Ser Tyr

1 5 10 15

Asp Ser Trp

<210>435

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>435

Cys Ala Thr Arg Thr Gly Tyr Ser Tyr Gly Phe Asn Phe Trp Ala Phe

1 5 10 15

Asp Ile Trp

<210>436

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>436

Cys Ala Thr Ser Pro Tyr Gly Val Phe Thr Leu Asp Tyr Trp

1 5 10

<210>437

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>437

Cys Ala Thr Val Thr Gly Tyr Ser Ser Ala Gly Ala Phe Asp Ile Trp

1 5 10 15

<210>438

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>438

Cys Ala Val Val Asp Asp Ala Phe Asp Ile Trp

1 5 10

<210>439

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>439

Cys Gly Ser Arg Gly Tyr Trp

1 5

<210>440

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>440

Cys His Ala Lys Gln Leu Arg Asn Gly Gln Met Tyr Thr Tyr Trp

1 5 10 15

<210>441

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>441

Cys His Ala Val Glu Asn Ile Leu Gly Arg Phe Val Asp Tyr Trp

1 5 10 15

<210>442

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>442

Cys His Gly Arg Asp Tyr Gly Ser Asn Ala Pro Gln Tyr Trp

1 5 10

<210>443

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>443

Cys Lys Ala Ala Pro Arg Trp Gly Gly Ala Thr Ala Tyr Trp

1 5 10

<210>444

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>444

Cys Lys Ile Tyr Gly Leu Asn Gly Gln Pro Leu Gly Ser Trp

1 5 10

<210>445

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>445

Cys Lys Leu Gln Val Arg Pro Ile Gly Tyr Ser Ser Ala Tyr Ser Arg

1 5 10 15

Asn Tyr Trp

<210>446

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>446

Cys Lys Gln His Pro Asn Gly Tyr Arg

1 5

<210>447

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>447

Cys Asn Ala Ala Ser Thr Val Thr Ala Trp Pro Tyr Tyr Gly Pro Asp

1 5 10 15

Tyr Trp

<210>448

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>448

Cys Asn Ala Asp Gly Tyr Ser Trp Asp Gly Arg Ser Gly Arg Arg Leu

1 5 10 15

Glu Leu Trp

<210>449

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>449

Cys Asn Ala Asp Ile Lys Thr Thr Thr Tyr Ser Pro Leu Arg Asn Tyr

1 5 10 15

Trp

<210>450

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>450

Cys Asn Ala Glu Thr Tyr Ser Gly Asn Thr Ile Trp

1 5 10

<210>451

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>451

Cys Asn Ala Phe Val Arg Ser Asp Phe Asp Arg Tyr Tyr Asp Tyr Trp

1 5 10 15

<210>452

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>452

Cys Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>453

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>453

Cys Asn Ala Asn Tyr Arg Gly Asn Arg Tyr Trp

1 5 10

<210>454

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>454

Cys Asn Ala Pro Ala Trp Leu Tyr Asp Asp Asp Tyr Trp

1 5 10

<210>455

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>455

Cys Asn Ala Val Thr Phe Gly Gly Asn Thr Ile Arg

1 5 10

<210>456

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>456

Cys Asn Ala Val Thr Tyr Asp Gly Tyr

1 5

<210>457

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>457

Cys Asn Ala Val Thr Tyr Asn Gly Tyr Thr Ile Arg

1 5 10

<210>458

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>458

Cys Asn Ala Val Val Val Gly Leu Ser Arg Arg Ile Asp Asn Ile Trp

1 5 10 15

<210>459

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>459

Cys Asn Lys Val Asn Ala Ile Thr Lys Leu

1 5 10

<210>460

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>460

Cys Asn Leu Arg Glu Trp Asn Asn Ser Gly Ala Gly Tyr Trp

1 5 10

<210>461

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>461

Cys Asn Thr Ser Pro Tyr Met His Asp Val Trp

1 5 10

<210>462

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>462

Cys Asn Thr Val Arg Pro Leu Trp Ala Trp

1 5 10

<210>463

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>463

Cys Asn Thr Tyr Pro Phe Pro Ile Tyr Lys Lys Gly Tyr Pro Phe Trp

1 5 10 15

<210>464

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>464

Cys Asn Val Asp Arg Thr Leu Tyr Gly Lys Tyr Lys Glu Tyr Trp

1 5 10 15

<210>465

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>465

Cys Ser Lys Gly Gly Val Tyr Gly Gly Thr Tyr Val Pro Asp Ser Trp

1 5 10 15

<210>466

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>466

Cys Ser Ser Arg Gly Tyr Trp

1 5

<210>467

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>467

Cys Thr Ala Gly Arg Ser Arg Tyr Leu Tyr Gly Ser Ser Leu Asn Gly

1 5 10 15

Pro Tyr Asp Tyr Trp

20

<210>468

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>468

Cys Thr Lys Gly Gly Ile Gln

1 5

<210>469

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>469

Cys Thr Leu Val Asn Glu Ile Lys Thr Trp Trp

1 5 10

<210>470

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>470

Cys Thr Arg Glu His Ser Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

1 5 10 15

<210>471

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>471

Cys Thr Arg Val Ala Trp Gly Leu Asp Tyr Trp

1 5 10

<210>472

<211>23

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>472

Cys Thr Thr Ala Gly Tyr Lys Ala Ala Arg Arg Ser Val Tyr Pro Arg

1 5 10 15

Ile Phe Asn Phe Asp Tyr Trp

20

<210>473

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>473

Cys Thr Thr Asp Asp Tyr Gly Asp Leu Thr His Leu Asp Tyr Trp

1 5 10 15

<210>474

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>474

Cys Thr Thr Asp Asp Tyr Gly Asp Gln Tyr Gly Met Asp Val Trp

1 5 10 15

<210>475

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>475

Cys Thr Thr Asp Leu Trp Asp Tyr Trp

1 5

<210>476

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>476

Cys Val Lys Asp Gly Gly Ser Phe Pro Leu Ala Tyr Ala Phe Asp Ile

1 5 10 15

Trp

<210>477

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>477

Cys Val Arg Gly Asp Ser Gly Trp Gly Ile Leu Tyr Tyr Val Met Asp

1 5 10 15

Val Trp

<210>478

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>478

Cys Val Arg Tyr Ala Trp Pro Glu Phe Asp His Trp

1 5 10

<210>479

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>479

Cys Val Tyr Gly Arg Asp Phe Asp Tyr Trp

1 5 10

<210>480

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>480

Cys Tyr Ala Asp Ser Arg Ser Ser Trp Tyr Asp Glu Tyr Leu Glu His

1 5 10 15

Trp

<210>481

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>481

Cys Tyr Ala Asn Ile Tyr Tyr Thr Arg Arg Ala Pro Glu Glu Tyr Trp

1 5 10 15

<210>482

<211>15

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>482

Cys Tyr Ala Arg Thr Gln Arg Met Gly Val Val Asn Ser Tyr Trp

1 5 10 15

<210>483

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>483

Cys Tyr Ala Arg Thr Val Ile Gly Gly Phe Gly Ala Phe Arg Ala His

1 5 10 15

Trp

<210>484

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>484

Cys Tyr Arg Arg Gln Trp Ala Ser Ser Trp Gly Ala Arg Asn Tyr Glu

1 5 10 15

Tyr Trp

<210>485

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>485

Cys Tyr Val Lys Leu Arg Asp Asp Asp Tyr Val Tyr Arg

1 5 10

<210>486

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>486

Gly Ala Ser Gln Ser Val Pro Arg Asn Ser Leu Ala

1 5 10

<210>487

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>487

His Gly Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210>488

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>488

Lys Ser Ser His Ser Leu Leu Ser Thr Ser Thr Asn Arg Asn His Leu

1 5 10 15

Ala

<210>489

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>489

Lys Ser Ser His Ser Leu Leu Ser Thr Ser Thr Asn Arg Asn Gln Leu

1 5 10 15

Ala

<210>490

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>490

Lys Ser Ser His Ser Leu Leu Tyr Ser Ser Asp Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210>491

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>491

Lys Ser Ser Gln Ser Ile Leu Ser Ser Ser Ser Asn Arg Asp Ser Leu

1 5 10 15

Ala

<210>492

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>492

Lys Ser Ser Gln Ser Val Leu Tyr Thr Thr Thr Asn Arg Asn His Ile

1 5 10 15

Ala

<210>493

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>493

Gln Ala Ser Gln Asp Ile Ala Asn Tyr Leu Asn

1 5 10

<210>494

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>494

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala

1 5 10

<210>495

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>495

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210>496

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>496

Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Ala

1 5 10

<210>497

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>497

Arg Ala Ser Gln Gly Ile Asn Ser Tyr Leu Ala

1 5 10

<210>498

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>498

Arg Ala Ser Gln Gly Ile Ser Asn Asn Leu Asn

1 5 10

<210>499

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>499

Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala

1 5 10

<210>500

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>500

Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210>501

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>501

Arg Ala Ser Gln Gly Ile Ser Arg Asp Leu Ala

1 5 10

<210>502

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>502

Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala

1 5 10

<210>503

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>503

Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala

1 5 10

<210>504

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>504

Arg Ala Ser Gln Asn Ile Gly Leu Tyr Leu Asn

1 5 10

<210>505

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>505

Arg Ala Ser Gln Arg Val Ser Asn Tyr Leu Asn

1 5 10

<210>506

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>506

Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn

1 5 10

<210>507

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>507

Arg Ala Ser Gln Ser Ile Ser Arg Trp Leu Ala

1 5 10

<210>508

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>508

Arg Ala Ser Gln Ser Ile Ser Arg Tyr Leu Asn

1 5 10

<210>509

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>509

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

1 5 10

<210>510

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>510

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>511

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>511

Arg Ala Ser Gln Ser Ile Glx Asn Tyr Leu Asn

1 5 10

<210>512

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>512

Arg Ala Ser Gln Ser Val Arg Ser Ser Asp Leu Ala

1 5 10

<210>513

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>513

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210>514

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>514

Arg Ala Ser Gln Ser Val Tyr Ser Asn Leu Ala

1 5 10

<210>515

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>515

Arg Ala Ser Gln Tyr Ile Ser Asn Tyr Leu Asn

1 5 10

<210>516

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>516

Arg Ala Ser Arg Asn Ile Asn Arg Tyr Leu Asn

1 5 10

<210>517

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>517

Arg Pro Ser Gln Ser Ile Gly Ser Trp Leu Ala

1 5 10

<210>518

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>518

Arg Ser Ser Gln Ser Leu Leu His Ser Asp Gly Tyr Thr Tyr Leu Tyr

1 5 10 15

<210>519

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>519

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210>520

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>520

Arg Val Ser Gln Gly Ile Ser Ser Tyr Leu Asn

1 5 10

<210>521

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>521

Arg Val Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210>522

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>522

Arg Glx Ser Gln Ser Glx Ser Glx Tyr Leu Asn

1 5 10

<210>523

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>523

Ser Gly Ser Ser Ser Asn Val Gly Asn Asn Tyr Val Ser

1 5 10

<210>524

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR1

<400>524

Trp Ala Ser Gln Ser Val Arg Gly Asn Tyr Val Ala

1 5 10

<210>525

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>525

Ala Ala Ser Ile Leu Gln Ser

1 5

<210>526

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>526

Ala Ala Ser Ser Leu His Ser

1 5

<210>527

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>527

Ala Ala Ser Ser Leu Leu Ser

1 5

<210>528

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>528

Ala Ala Ser Ser Leu Gln Gly

1 5

<210>529

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>529

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>530

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>530

Ala Ala Ser Thr Leu Glu Ser

1 5

<210>531

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>531

Ala Ala Ser Thr Leu Gln Arg

1 5

<210>532

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>532

Ala Ala Ser Thr Leu Gln Ser

1 5

<210>533

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>533

Asp Ala Lys Gly Leu His Pro

1 5

<210>534

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>534

Asp Ala Ser Asn Leu Glu Thr

1 5

<210>535

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>535

Asp Ala Ser Asn Leu Gln Ser

1 5

<210>536

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>536

Asp Ala Ser Asn Arg Ala Ala

1 5

<210>537

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>537

Asp Ala Ser Asn Arg Ala Gly

1 5

<210>538

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>538

Asp Ala Ser Asn Arg Gln Ser

1 5

<210>539

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>539

Asp Ala Ser Ser Leu Glu Ser

1 5

<210>540

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>540

Asp Ala Ser Ser Leu Gln Arg

1 5

<210>541

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>541

Asp Asn Asp Lys Arg Pro Ser

1 5

<210>542

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>542

Asp Thr Ser Asn Arg Ala Thr

1 5

<210>543

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>543

Gly Ala Ser Gln Arg Ala Thr

1 5

<210>544

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>544

Gly Ala Ser Thr Arg Ala Thr

1 5

<210>545

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>545

Gly Ser Ser Ser Arg Ala Thr

1 5

<210>546

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>546

Gly Ser Ser Thr Leu Gln Ser

1 5

<210>547

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>547

Gly Thr Ser Asn Leu Gln Ser

1 5

<210>548

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>548

Gly Thr Ser Ser Leu His Thr

1 5

<210>549

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>549

Lys Ala Ser Ser Leu Glu Ser

1 5

<210>550

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>550

Leu Gly Ser Asn Arg Ala Ser

1 5

<210>551

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>551

Ser Ala Ser Ser Leu Gln Ser

1 5

<210>552

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>552

Ser Ala Ser Thr Leu Gln Ser

1 5

<210>553

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>553

Thr Leu Ser Tyr Arg Ala Ser

1 5

<210>554

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>554

Trp Ala Ser Ser Arg Lys Ser

1 5

<210>555

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>555

Glx Ala Ser Ser Leu Gln Ser

1 5

<210>556

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR2

<400>556

Glx Ala Ser Thr Leu Glu Ser

1 5

<210>557

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>557

Cys Glu Ser Trp Asp Ser Ser Leu Ser Ser Glu Val Phe

1 5 10

<210>558

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>558

Cys Leu Gln Asp Phe Ser Phe Pro Trp Thr Phe

1 5 10

<210>559

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>559

Cys Leu Gln Asp Tyr Ser Tyr Pro Arg Thr Phe

1 5 10

<210>560

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>560

Cys Leu Gln Asp Tyr Thr Asp Pro Phe Thr Phe

1 5 10

<210>561

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>561

Cys Leu Gln His Asn Ser Tyr Pro Phe Thr Phe

1 5 10

<210>562

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>562

Cys Met Gln Ala Leu Glu Ala Leu Phe Thr Phe

1 5 10

<210>563

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>563

Cys Met Gln Ala Leu Gln Thr Pro Ile Thr Phe

1 510

<210>564

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>564

Cys Met Gln Ala Thr Gln Phe Pro Leu Thr Phe

1 5 10

<210>565

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>565

Cys Gln His Arg Ser Asn Trp Pro Leu Thr Phe

1 5 10

<210>566

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>566

Cys Gln Gln Ala Asn Ser Phe Pro Phe Thr Phe

1 5 10

<210>567

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>567

Cys Gln Gln Ala Asn Ser Leu Phe Thr Phe

1 5 10

<210>568

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>568

Cys Gln Gln Ala Tyr Ser Phe Pro Trp Thr Phe

1 5 10

<210>569

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>569

Cys Gln Gln Phe Asp Arg Ser Pro Leu Thr Phe

1 5 10

<210>570

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>570

Cys Gln Gln Gly Asn Ser Phe Pro Tyr Thr Phe

1 5 10

<210>571

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>571

Cys Gln Gln Gly Tyr Gly Thr Pro Pro Met Phe

1 5 10

<210>572

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>572

Cys Gln Gln Gly Tyr Asn Thr Pro Arg Thr Phe

1 5 10

<210>573

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>573

Cys Gln Gln Ile His Ser Tyr Pro Leu Thr Phe

1 5 10

<210>574

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>574

Cys Gln Gln Ser Phe Ser Thr Pro Arg Thr Phe

1 5 10

<210>575

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>575

Cys Gln Gln Ser Asn Ser Phe Pro Tyr Thr Phe

1 5 10

<210>576

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>576

Cys Gln Gln Ser Ser Ser Thr Pro Tyr Thr Phe

1 5 10

<210>577

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>577

Cys Gln Gln Ser Tyr Asp Ser Pro Trp Thr Phe

1 5 10

<210>578

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>578

Cys Gln Gln Ser Tyr Ile Thr Pro Leu Thr Phe

1 5 10

<210>579

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>579

Cys Gln Gln Ser Tyr Asn Thr Pro Arg Thr Phe

1 5 10

<210>580

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>580

Cys Gln Gln Ser Tyr Asn Val Pro Phe Thr Phe

1 5 10

<210>581

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>581

Cys Gln Gln Ser Tyr Arg Ile His Trp Thr Phe

1 5 10

<210>582

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>582

Cys Gln Gln Ser Tyr Arg Thr Pro Leu Thr Phe

1 5 10

<210>583

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>583

Cys Gln Gln Ser Tyr Arg Tyr Pro Thr Phe

1 5 10

<210>584

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>584

Cys Gln Gln Ser Tyr Ser Ala Pro Leu Ser Phe

1 5 10

<210>585

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>585

Cys Gln Gln Ser Tyr Ser Ala Pro Leu Thr Phe

1 5 10

<210>586

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>586

Cys Gln Gln Ser Tyr Ser Phe Pro Pro Phe Thr Phe

1 5 10

<210>587

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>587

Cys Gln Gln Ser Tyr Ser Ile Pro Phe Thr Phe

1 5 10

<210>588

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>588

Cys Gln Gln Ser Tyr Ser Leu Pro Leu Thr Phe

1 5 10

<210>589

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>589

Cys Gln Gln Ser Tyr Ser MetPro Leu Thr Phe

1 5 10

<210>590

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>590

Cys Gln Gln Ser Tyr Ser Pro Pro Phe Thr Phe

1 5 10

<210>591

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>591

Cys Gln Gln Ser Tyr Ser Ser Pro Pro Thr Phe

1 5 10

<210>592

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>592

Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe

1 5 10

<210>593

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>593

Cys Gln Gln Ser Tyr Ser Thr Pro Trp Thr Phe

1 5 10

<210>594

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>594

Cys Gln Gln Ser Tyr Ser Thr Pro Tyr Thr Phe

1 5 10

<210>595

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>595

Cys Gln Gln Ser Tyr Ser Thr Ser Ile Thr Phe

1 5 10

<210>596

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>596

Cys Gln Gln Ser Tyr Ser Val Pro Tyr Thr Phe

1 5 10

<210>597

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>597

Cys Gln Gln Ser Tyr Thr Ser Arg Leu Thr Phe

1 5 10

<210>598

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>598

Cys Gln Gln Ser Tyr Thr Thr Pro Phe Thr Phe

1 5 10

<210>599

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>599

Cys Gln Gln Thr Tyr Ser Ile Pro Pro Thr Phe

1 5 10

<210>600

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>600

Cys Gln Gln Thr Tyr Thr Ile Pro Phe Thr Phe

1 5 10

<210>601

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>601

Cys Gln Gln Tyr Gly Arg Ser Pro Arg Tyr Ser Phe

1 5 10

<210>602

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>602

Cys Gln Gln Tyr His Asn Trp Pro Pro Glu Tyr Thr Phe

1 5 10

<210>603

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>603

Cys Gln Gln Tyr Asn Asn Trp Pro Pro Ile Thr Phe

1 5 10

<210>604

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>604

Cys Gln Gln Tyr Asn Asn Trp Pro Tyr Thr Phe

1 5 10

<210>605

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>605

Cys Gln Gln Tyr Tyr Asn Ile Pro Tyr Ser Phe

1 510

<210>606

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>606

Cys Gln Gln Tyr Tyr Ser Thr Pro Gln Thr Phe

1 5 10

<210>607

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-light CDR3

<400>607

Cys Gln Gln Tyr Tyr Ser Thr Pro Tyr Thr Phe

1 5 10

<210>608

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>608

Cys Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>609

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>609

Cys Asn Ala Ala Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>610

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>610

Cys Asn Ala Lys Ala Pro Trp Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>611

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>611

Cys Asn Ala Lys Arg Ala Trp Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>612

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>612

Cys Asn Ala Lys Arg Pro Ala Gly Ser Arg Asp Glu Tyr Trp

1 5 10

<210>613

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>613

Cys Asn Ala Lys Arg Pro Trp Ala Ser Arg Asp Glu Tyr Trp

1 5 10

<210>614

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>614

Cys Asn Ala Lys Arg Pro Trp Gly Ala Arg Asp Glu Tyr Trp

1 5 10

<210>615

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>615

Cys Asn Ala Lys Arg Pro Trp Gly Ser Ala Asp Glu Tyr Trp

1 5 10

<210>616

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>616

Cys Asn Ala Lys Arg Pro Trp Gly Ser Arg Ala Glu Tyr Trp

1 5 10

<210>617

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>617

Cys Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Ala Tyr Trp

1 5 10

<210>618

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic molecular recognition Unit-heavy CDR3

<400>618

Cys Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Ala Trp

1 5 10

<210>619

<211>648

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation of an-LRP 6E3E4_ O75581 construct in the laboratory

<400>619

Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala Asp Ile Arg Arg Ile Ser

1 5 10 15

Leu Glu Thr Asn Asn Asn Asn Val Ala Ile Pro Leu Thr Gly Val Lys

20 25 30

Glu Ala Ser Ala Leu Asp Phe Asp Val Thr Asp Asn Arg Ile Tyr Trp

35 40 45

Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg Ala Phe Met Asn Gly Ser

50 55 60

Ala Leu Glu His Val Val Glu Phe Gly Leu Asp Tyr Pro Glu Gly Met

65 70 75 80

Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr Trp Ala Asp Thr Gly Thr

85 90 95

Asn Arg Ile Glu Val Ser Lys Leu Asp Gly Gln His Arg Gln Val Leu

100 105 110

Val Trp Lys Asp Leu Asp Ser Pro Arg Ala Leu Ala Leu Asp Pro Ala

115 120 125

Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly Gly Lys Pro Lys Ile Asp

130 135 140

Arg Ala Ala Met Asp Gly Ser Glu Arg Thr Thr Leu Val Pro Asn Val

145 150 155 160

Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr Ala Lys Arg Arg Leu Tyr

165 170 175

Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu Ser Ser Asn Met Leu Gly

180 185 190

Leu Asn Arg Glu Val Ile Ala Asp Asp Leu Pro His Pro Phe Gly Leu

195 200 205

Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr Asp Trp Ser Arg Arg Ser

210 215 220

Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln Asn Arg Thr Ile Ile Gln

225 230 235 240

Gly His Leu Asp Tyr Val Met Asp Ile Leu Val Phe His Ser Ser Arg

245 250 255

Gln Ser Gly Trp Asn Glu Cys Ala Ser Ser Asn Gly His Cys Ser His

260 265 270

Leu Cys Leu Ala Val Pro Val Gly Gly Phe Val Cys Gly Cys Pro Ala

275 280 285

His Tyr Ser Leu Asn Ala Asp Asn Arg Thr Cys Ser Ala Pro Thr Thr

290 295 300

Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile Asn Arg Met Val Ile Asp

305 310 315 320

Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro Ile His Ser Leu Arg Asn

325 330 335

Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp Lys Gln Leu Tyr Trp Ile

340 345 350

Asp Ser Arg Gln Asn Met Ile Arg Lys Ala Gln Glu Asp Gly Ser Gln

355 360 365

Gly Phe Thr Val Val Val Ser Ser Val Pro Ser Gln Asn Leu Glu Ile

370 375 380

Gln Pro Tyr Asp Leu Ser Ile Asp Ile Tyr Ser Arg Tyr Ile Tyr Trp

385 390 395 400

Thr Cys Glu Ala Thr Asn Val Ile Asn Val Thr Arg Leu Asp Gly Arg

405 410 415

Ser Val Gly Val Val Leu Lys Gly Glu Gln Asp Arg Pro Arg Ala Val

420 425 430

Val Val Asn Pro Glu Lys Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu

435 440 445

Arg Ser Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Thr Glu Arg Glu

450 455 460

Val Leu Phe Phe Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp

465 470 475 480

Ser Arg Leu Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile

485 490 495

Glu Ser Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser

500 505 510

Asn Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr

515 520 525

Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr Gly

530 535 540

Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu Ser Asp

545 550 555 560

Ile His Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg Gln His Pro

565 570 575

Cys Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys Leu Val Lys Gly

580585 590

Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His Leu Val Leu Leu Gln

595 600 605

Asp Glu Leu Ser Cys Gly Glu Pro Pro Ser Gly Ser Gly Gly Leu Asn

610 615 620

Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Ser Gly Ser

625 630 635 640

His His His His His His His His

645

<210>620

<211>130

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation of-Lrp 6E3E4 VHH26 Complex in the laboratory

<400>620

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Gly Ser Gly Ser Gly His His His His

115 120 125

His His

130

<210>621

<211>122

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> preparation of-Lrp 6E3E4 VHH36 Complex in the laboratory

<400>621

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Ile Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Gly Ile Arg Trp Ser Gly Gly Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Ile Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly

85 90 95

Ser Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

100 105 110

Ser Gly Ser Gly His His His His His His

115 120

Claims (34)

1. An isolated antibody or antigen binding fragment thereof that binds to one or more LRP5 or LRP6 receptors, comprising a sequence comprising:

(i) CDRH1, CDRH2 and CDRH3 sequences as shown for any of the antibodies of table 1A;

(ii) CDRL1, CDRL2 and CDRL3 sequences as shown for any of the antibodies of table 1A; and/or

(iii) The CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences shown for any of the antibodies of Table 2,

or a variant of said antibody or antigen-binding fragment thereof, said variant comprising one or more amino acid modifications, wherein said variant comprises fewer than 8 amino acid substitutions in said CDR sequences.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs 1-24.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs 1-24.

4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is humanized.

5. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is a single chain antibody, scFv, monovalent antibody lacking a hinge region, VHH, or sdAb, or minibody.

6. The isolated antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof is a VHH VHH or a sdAb.

7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a Fab or Fab' fragment.

8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof is a fusion protein.

9. The isolated antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof is fused to a polypeptide sequence that binds one or more frizzled (Fzd) receptors.

10. The isolated antibody or antigen-binding fragment thereof of claim 9, wherein the polypeptide sequence that binds one or more Fzd receptors is an antibody or antigen-binding fragment thereof that binds one or more Fzd receptors.

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof binds to LRP 5.

12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment thereof binds to LRP 6.

13. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the antibody or antigen-binding fragment thereof binds to: LRP5 and LRP 6.

14. An isolated antibody or antigen-binding fragment thereof that competes for binding to human LRP5 or LRP6 with the antibody of any one of claims 1-13.

15. The isolated antibody or antigen binding fragment thereof of any one of claims 1-14, which binds to the LRP5 or LRP6 with a KD of 50 μ Μ or lower.

16. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 15, which modulates the Wnt signaling pathway in a cell, optionally a mammalian cell.

17. The isolated antibody or antigen-binding fragment thereof of claim 16, which increases signaling through the Wnt signaling pathway in the cell.

18. The isolated antibody or antigen-binding fragment thereof of claim 16, which reduces signaling through the Wnt signaling pathway in the cell.

19. The isolated antibody or antigen-binding fragment thereof of any one of claims 16 to 18, wherein the Wnt signaling pathway is a canonical Wnt signaling pathway.

20. The isolated antibody or antigen-binding fragment thereof of any one of claims 16 to 18, wherein the Wnt signaling pathway is an atypical Wnt signaling pathway.

21. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 20.

22. An expression vector comprising the isolated polynucleotide of claim 22.

23. An isolated host cell comprising the expression vector of claim 22.

24. A pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier and a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-20 or 33.

25. A method for agonizing the Wnt signaling pathway in a cell, the method comprising contacting the cell with the isolated antibody or antigen-binding fragment thereof of claim 17.

26. The method of claim 24, wherein the antibody or antigen-binding fragment thereof is a fusion protein comprising a polypeptide sequence that binds to one or more frizzled (Fzd) receptors.

27. A method for inhibiting a Wnt signaling pathway in a cell, the method comprising contacting the cell with the isolated antibody or antigen-binding fragment thereof of claim 18.

28. A method for treating a subject having a disease or disorder associated with reduced Wnt signaling, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 24, wherein the isolated antibody or antigen-binding fragment thereof is an agonist of the Wnt signaling pathway.

29. The method of claim 28, wherein the disease or disorder is selected from the group consisting of: bone fracture, stress fracture, vertebral compression fracture, osteoarthritis, osteoporosis, osteoporotic fracture, nonunion fracture, delayed union fracture, spinal fusion, preoperative optimization of spinal surgery, osteonecrosis, osteointegration of implants or orthopedic devices, osteogenesis imperfecta, bone graft, tendon repair, tendon-bone integration, tooth growth and regeneration, maxillofacial surgery, dental implant, periodontal disease, maxillofacial reconstruction, jaw, hip, or femoral head necrosis, avascular necrosis, hair loss, hearing loss, vestibular dysfunction, macular degeneration, age-related macular degeneration (AMD), vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs 'dystrophe, corneal diseases, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, sarcopenia and cachexia, diseases affecting the Blood Brain Barrier (BBB), spinal cord injury, spinal cord disease, oral mucositis, short bowel syndrome, Inflammatory Bowel Disease (IBD), metabolic syndrome, diabetes, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency, wound healing, diabetic foot ulcer, bedsores, venous leg ulcers, bullous epidermolysis, skin aplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell disorders, immunodeficiency, graft-versus-host disease, acute kidney injury, chronic kidney disease, Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis, acute liver failure of all causes, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of all causes, liver cirrhosis, liver fibrosis of all causes, portal hypertension, chronic pulmonary fibrosis, acute liver failure of all causes, chronic liver fibrosis of all causes, chronic liver disease of all causes, chronic liver disease of all, Chronic hepatic insufficiency of all causes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis c virus-induced liver disease (HCV), hepatitis b virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis a virus-induced liver disease (HAV) and hepatitis d virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small liver (small liver) syndrome in liver surgery and transplantation, congenital liver diseases and disorders, any other liver disorder or disease caused by genetic disease, degeneration, aging, drug or injury.

30. A method for treating a subject having a disease or disorder associated with increased or enhanced Wnt signaling, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 24, wherein the isolated antibody or antigen-binding fragment thereof is an inhibitor of the Wnt signaling pathway.

31. The method of claim 29, wherein the disease or disorder is selected from the group consisting of: tumors and cancers, degenerative disorders, fibrosis of any organ or tissue, heart failure, coronary artery disease, ectopic ossification, osteopetrosis, congenital high bone mass disorders.

32. An isolated antibody or antigen-binding fragment thereof that binds to the LRP6 receptor, wherein the antibody or antigen-binding fragment thereof binds to the E3 β -propeller region of the LRP6 receptor or a corresponding region of the LRP5 receptor.

33. An isolated antibody or antigen binding fragment thereof that binds to the LRP6 receptor, wherein the antibody or antigen binding fragment thereof binds to one or more epitopes within the region of the LRP6 receptor comprising or consisting of amino acid residues 637-878 or the corresponding region of the LRP5 receptor.

34. An isolated antibody or antigen-binding fragment thereof that binds to one or more LRP receptors, wherein the antibody or antigen-binding fragment thereof contacts the LRP receptor at any one of the sets of amino acid residues indicated in Table 3 at a distance of less than 5 angstroms.

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